JP4825383B2 - Method for cloning and producing MseI restriction endonuclease - Google Patents

Description

【００７４】
ＢａｍＨＩ、ＭｕｎＩ及びＢａｎＩＩで上記プラスミドを消化し、構成プロモータを含有するベクターの骨格をゲルで精製した（類似する大きさのプラスミド断片を除去することによってベクター骨格のゲルによる精製を助けるために、ＢａｎＩＩ消化を含めた）。
【００７５】
上述の構成プロモータの下流に挿入するのにＭｓｅＩメチラーゼ遺伝子を調製するために、ベント（登録商標名）ＤＮＡポリメラーゼ、１ｘサーモポル緩衝液、４ｍＭのＭｇＳＯ_４、１００μｌのＰＣＲ反応物における鋳型としてＭｓｅＩメチラーゼ遺伝子を含有する８０ｎｇのｐＶＲ１９プラスミド（Ｒ．Ｖａｉｓｖｉｌａ）、及びメチラーゼ遺伝子に関してＢａｍＨＩ部位の上流に導入する、５’−ＧＡＡＣＣＧＧＡＴＣＣＧＡＣＣＣＴＧＡＧＴＧＡＧＡＡＣＡＴＧＣＣ−３’（ＳＥＱ ＩＤ ＮＯ：１５）プライマー並びにＭｆｅＩ部位の下流に導入する５’−ＡＧＧＴＣＧＣＡＡＴＴＧＣＣＡＧＧＧＧＴＣＧＴＣＴＴＣＡＣＴＣＧＣＴＡＣ−３’（ＳＥＱ ＩＤ ＮＯ：１６）プライマーを用いてＰＣＲを行った。９５℃にて１０秒間、６０℃にて６０秒間及び７２℃にて７５秒間を２５サイクル行った。キアクイックＰＣＲ精製プロトコールを用いて、得られた１０１９塩基対のＰＣＲ産物を精製し、ＢａｍＨＩとＭｕｎＩで順に消化し、キアクイックＰＣＲ精製プロトコールを用いてもう１回精製した。
【００７６】
４つのＢａｍＨＩ−ＭｕｎＩベクター骨格すべてにＭｓｅＩメチラーゼ遺伝子を連結し、ＥＲ２６８８細胞を形質転換して、ルリア−ベルターニ（Ｌｕｒｉａ−Ｂｅｒｔａｎｉ）（リットル当り１グラムのグルコースと１グラムのＭｇＣｌ_２を補った；以下補完ＬＢという）寒天プレートに播いた。しかしながら、おそらく細胞における高いレベルのメチル化に由来する不安定性のために、発現の最も高い２つのレベルのもとにＭｓｅＩメチラーゼを配置するという試みは失敗した。さらに低い２つのレベルの発現を含有するコンストラクト、ｐＮＫＲ１７０７ＭｓｅＩｍ及びｐＮＫＲ２２１３ＭｓｅＩｍは、このような細胞から精製したプラスミドＤＮＡのＭｓｅＩによる制限（５０μｌ容量における１μｇのプラスミドＤＮＡ、２０単位のＭｓｅＩで３７℃にて１時間）に対する感受性により判断されるように、細胞性ＤＮＡの完全なメチル化は生じなかった。
【００７７】
（２)エラー傾向ＰＣＲによる無作為に変異誘発を行った構成プロモータのライブラリの構築
ｐＮＫＲ２２１３ＭｓｅＩｍと明らかに不安定なｐＮＫＲ１７８６ＭｓｅＩｍとの間の、ＭｓｅＩメチラーゼに対する中間レベルのプロモータコンストラクトを見つけるために、構成プロモータ領域を無作為ＰＣＲ変異誘発と選定の対象とした。変異誘発プロトコールでは、高レベルのＴａｑＤＮＡポリメラーゼ（１００μｌの反応容量当り５単位）、不均等ｄＮＴＰプール（１．２ｍＭのｄＣＴＰとＴＴＰ；０．２ｍＭのｄＡＴＰとｄＧＴＰ）、高レベルのＭｇＣｌ２（７ｍＭ）、ＭｎＣｌ２（０．５ｍＭ）の存在、１００μｌ容量当り２ｎｇのｐＮＫＲ１７０７ＭｓｅＩｍ及びサイクル数の多い（３５）ＰＣＲを採用した。ＡｇｅＩ制限部位及びＢａｍＨＩ制限部位でＭｓｅＩメチラーゼに隣接するプライマーはそれぞれ、５’−ＧＣＧＡＴＡＣＡＧＡＣＣＧＧＴＴＣＡＧＡＣＡＧＧＡＴＡＡＡＧ−３’（ＳＥＱ ＩＤ ＮＯ：１７）及び５’−ＧＧＴＣＧＧＡＴＣＣＧＧＣＧＡＴＡＣＡＧＣＧＡＧ−３’（ＳＥＱ ＩＤ ＮＯ：１８）。
【００７８】
ＰＣＲの後、ＡｇｅＩ及びＢａｍＨＩで変異誘発したプロモータコピーを切断し、キアゲンのゲル精製キットでゲルによる精製を行い、その内因性構成プロモータを精製して除いた、ＡｇｅＩ−ＢａｍＨＩ制限部位を持つｐＮＫＲＭｓｅＩｍコンストラクトに連結した。ＥＲ２６８８コンピテント細胞にエレクトロポレーションした後、２０，０００個のコロニーを得た。このようなコロニーをプールし、キアゲン精製プロトコールを用いてプラスミドを精製した。これによって、ＭｓｅＩメチラーゼ遺伝子の上流に無作為で変異誘発された構成プロモータのライブラリが構築された。
【００７９】
（３）ＭｓｅＩ制限切断に耐性のプラスミドをもたらすクローンの選定
安定な、高レベルのメチル化を生じる変異誘発された構成プロモータを処理するプラスミドを選定するために、５μｇのプラスミドライブラリをＭｓｅＩ制限（５μｇのＤＮＡ、５０単位のＭｓｅＩ）に３７℃で４時間暴露し、続いて６５℃で２０分インキュベートしてＭｓｅＩ制限エンドヌクレアーゼを不活化した。暴露したプールの一部（２５０ｎｇ）でＥＲ２６８８カルシウム−コンピテント細胞を形質転換し、補完ＬＢ寒天プレートに播いて、３７℃にて一晩増殖させた。これによって６３コロニーが生じた。
【００８０】
６３コロニーのうち６個を無作為に選択し、さらに個々の実験を行った；１０ｍｌの補完ＬＢ培地中での一晩の増殖の後、キアゲンのキア−プレップスピン・ミニプレップ・プロトコールを用いてプラスミドＤＮＡを精製した。１００ｎｇの精製プラスミドＤＮＡを３７℃にて３０分間２０単位のＭｓｅＩに暴露すると、６個すべて完全に制限されたということは、不適当なレベルのメチル化を示している。
【００８１】
残りの５７コロニーをプールし、キアゲンのプラスミド精製プロトコールを用いてプラスミドの精製を行った。このプラスミドのプールから、５０ｎｇをさらに長い（一晩）５０単位のＭｓｅＩ暴露の対象とし、続いて６５℃にて２０分間インキュベートしてＭｓｅＩの制限エンドヌクレアーゼを不活化した。暴露したプールの一部（４ｎｇ）でＥＲ２６８８カルシウム−コンピテント細胞を形質転換し、補完ＬＢ寒天プレートに播いて、３７℃にて一晩増殖させた。これによって１３コロニーが生じた。
【００８２】
１３コロニーのうち９個を無作為に選択し、さらに個々の実験を行った；一晩の増殖及び以前記載したようなプラスミドの精製の後、１μｇのプラスミドＤＮＡを５０μｌの反応容量中の５０単位のＭｓｅＩとともに３７℃にて一晩インキュベートすると、９個のうち７個で完全にメチル化されたことが判明した。
【００８３】
細胞に存在するメチル化のレベルをさらに立証するために、培養の対数増殖期の間にプラスミド精製のために７コロニーを回収した（一晩の培養を新鮮な補完ＬＢ増殖培地で１：１００に希釈した４時間後、３７℃にて、細胞を回収した。）。かかる細胞は、発現されたＭｓｅＩメチラーゼによるメチル化が完全なメチル化を達成することができないような速度でＤＮＡを複製していることが予想される。キアゲンのキア−プレップ精製プロトコールを用いて、このような対数的に増殖している培養からプラスミドＤＮＡを精製し、０．５μｇのこのプラスミドＤＮＡを３７℃にて一晩、５０単位のＭｓｅＩとインキュベートした。このさらにメチル化が困難な基準を用いて、７コロニーのうち３個が完全に保護され（メチル化され）、制限切断に抵抗性であった。
【００８４】
安定で、完全なレベルのＭｓｅＩメチル化を生じる３個のクローン（＃４、＃９及び＃１０）は、ＡｇｅＩ及びＢａｍＨＩでマッピングすることによって、及びプロモータ領域の上流にアニーリング部位を持つプライマーを用いて配列決定することによって調べられたプロモータ領域を有していた（５’−ＧＧＡＴＣＴＴＣＣＡＧＴＧＧＴＧＣＡＴＧＡＡＣＧ−３’（ＳＥＱ ＩＤ ＮＯ：１９））。３クローンのうち２個（＃９と＃１０）は同一であり；従って、記載された２段階選定工程によって、結果として、安定な、ＭｓｅＩメチル化の完全なレベルが得られる２つの別々のプロモータが見い出された。
【００８５】
思いがけないことに、両プロモータ＃４及び＃９／＃１０は、実験設計したような変異誘発された構成プロモータではなかったが、その代わり、プラスミド調製物に存在する低レベルの大腸菌ＤＮＡの混入に起源があるに違いないＡｇｅＩ−ＢａｍＨＩ大腸菌配列であった。
【００８６】
＃４プロモータは、ＡｇｅＩ／ＢａｍＨＩのマッピングによれば、約１０００塩基対の長さであると思われ、配列決定によれば、最初の４３８塩基対は、大腸菌Ｋ−１２ＭＧ１６５５区分３４９（寄託受入番号、第ＡＥ０００４５９号）の塩基番号７８１３〜８２５１と同一であった。配列データを調べると、ＢａｍＨＩ部位は、塩基番号８８１４で見い出され、それによって１００２塩基対のＡｇｅＩ−ＢａｍＨＩ大腸菌断片が得られた。この大腸菌配列は、ｙｉｇＷ＿２ｏｒｆの５’末端及び２つの予測されたプロモータを含有しており、そのうちの１つは、ＭｓｅＩメチラーゼと同一方向を向いている（８６７２〜８７０４番目）。
【００８７】
＃９／＃１０プロモータのマッピングは、ＡｇｅＩ／ＢａｍＨＩによって約４２０塩基対の長さであると思われ；配列決定によれば、プロモータは大腸菌Ｋ−１２ＭＧ１６５５の区分４１（寄託受入番号、第ＡＥ０００１５１号）の塩基番号２５１１〜２９９８と同一であった。これによって４８８塩基対のＡｇｅＩ−ＢａｍＨＩ大腸菌断片が決定され、それは、ｃｏｆ ｏｒｆの５’末端及び塩基番号２６０５〜２６３２及び２７１４〜２７４２にてＭｓｅＩメチラーゼと同じ方向を向いた２つの予測されたプロモータを含有する。この＃９／＃１０の配列を用いてさらに調べた。
【００８８】
（４）ＭｓｅＩメチラーゼ発現のさらなる最適化
上記の戦略を用いて、プラスミドｐＶＲ−２５でＭｓｅＩエンドヌクレアーゼの発現を許すＭｓｅＩメチルトランスフェラーゼの発現レベルを達成した。思いがけないことに、初めて形質転換され、増殖させた場合、プラスミドｐＶＲ−２５の中で最適化されたＭｓｅＩメチラーゼ（上記＃９）及びＭｓｅＩエンドヌクレアーゼを持つＥＲ２５６６宿主細胞はＭｓｅＩエンドヌクレアーゼを発現したが、このコンストラクトをグリセロール中で−７０℃にて保存した場合、ＭｓｅＩは安定的に維持されなかった。
【００８９】
宿主のＭｓｅＩのさらに大きな修飾を達成するためにＭｓｅＩメチラーゼのコンストラクトをさらに修飾した。上述したように、おそらく細胞における高いレベルのメチル化に由来する不安定性のために、最も高い２つのレベルの構成的な発現のもとにＭｓｅＩメチラーゼを配置するという試みは失敗した。最大限、認容されるレベルのメチル化を達成するために、新しいＭ．ＭｓｅＩ発現プラスミド、ｐＶＲ−２６を構築した。上記（３）で記載されたものに由来する第２のプロモータを挿入することによってｐＶＲ−２６を構築した（表２を参照のこと）。Ｍ．ＭｓｅＩをコードする領域（ｍｓｅＩＭ遺伝子）及びプラスミドｐＮＫＲ１７０７ｍｓｅＩＭ−９（ＰｍｅＩとＭｆｅＩで消化された）からの上流のプロモータを含有する１．２４４−ｋｂのＤＮＡ断片を切断し、ＥｃｏＲＩとＨｉｎｃＩＩで切断したベクターｐＮＥＢ１９３（米国マサチューセッツ州、ベバリー、ニューイングランド・バイオラボズ社）におけるＰ_{ｌａｃＵＶ５}プロモータのすぐ下流に挿入することによって、これを行った。もう１つのＭｓｅＩメチラーゼのコンストラクト、ｐＶＲ２７は、Ｐ_{ｌａｃＵＶ５}プロモータとｐＶＲ２６からのｌａｃＩオペレータを含有する０．３７９−ｋｂのＰｍｅＩ−ＡｆｌＩＩＩ断片を削除することによって作成した。ｐＶＲ−２６ｍｓｅＩＭメチラーゼ発現ベクターによってＭｓｅＩエンドヌクレアーゼの安定な発現が得られた。
【００９０】
【表２】

【００９１】
実施例５：ＭｓｅＩ制限エンドヌクレアーゼ発現の最適化
（１）発現ベクターの構築
当該技術で周知のように、制限エンドヌクレアーゼは細胞毒性のタンパク質である。多くの場合、高い発現を助長にするように設計されたプラスミドに毒性遺伝子をクローニングする試みは極めて難しい。細胞毒性遺伝子を発現するのに特に好ましいプラスミドの１つは、ｐＬＴ７Ｋである（Kong, et al., Nucl. Acids Res., 28:3216-3222, 2000）。このプラスミドは、複製機能をコードする断片（ｏｒｉ）、β−ラクタマーゼをコードする遺伝子、及びクローニングに好適な制限エンドヌクレアーゼに隣接するカナマイシン耐性をコードする遺伝子を含有する。ｃＩ８５７遺伝子は、ＰＬとＰＲ（ラムダバクテリオファージの主要な、それぞれ左側と右側のプロモータ）を重ね合わせるＤＮＡ配列（ＣＩオペレータ）に条件付きで結合するラムダバクテリオファージのリプレッサタンパク質に由来する変異体をコードする。ｌａｃＩ遺伝子は、リプレッサタンパク質ＬａｃＩをコードし、それは、ＰＴ７（バクテリオファージＴ７のＲＮＡポリメラーゼ転写プロモータ）を重ね合わせるように構築されたＤＮＡ配列（ｌａｃオペレータ）に条件付きで結合する。簡単に言えば、ＩＰＴＧ無しでの高温（４２℃）では、アンチセンスのプロモータが活性を持ち、Ｐ_Ｔ７はＬａｃＩによって抑制される。ＩＰＴＧを伴った３０℃では、発現はＰ_Ｔ７から生じる。
【００９２】
ＭｓｅＩ制限エンドヌクレアーゼ遺伝子の過剰発現にｐＬＴ７Ｋを適合させるために、ＮｄｅＩ制限エンドヌクレアーゼ部位とリボソーム結合部位を導入した。さらに、ｃｏｌＥＩレプリコンをｐ１５Ａレプリコンに変更し、コピー数を１／３に減らした（約５０から約１５に）。これを達成するために、ＡｃｌＩとＢａｍＨＩでｐＬＴ７Ｋを消化した。ｃＩ８５７、ラムダＰＬ、Ｋｎ耐性遺伝子及びＴ７プロモータを含有する得られた１．２−ｋｂの断片をキアゲンのキアクイック・ゲル精製キット（カタログ番号２８７０４）を用いて寒天ゲルから単離し、予めＣｌａＩとＢａｍＨＩで消化したｐＡＣＹＣ１８４−Ｔ７ｔｅｒΔＰｓｈＡＩベクターに連結した。ｐＡＣＹＣ１８４−Ｔ７ｔｅｒΔＰｓｈＡＩは、ｐＡＣＹＣ１８４−Ｔ７ｔｅｒのＰｓｈＡＩ欠失誘導体である。このコンストラクトをｐＶＲ−２４と命名した（図１４）。
【００９３】
正方向（５’−ＡＧＡＣＴＣＣＣＣＣＡＴＡＴＧＡＣＣＣＡＣＧＡＡＣＣＧＡＣＧＧＡＴＧ−３’）（ＳＥＱ ＩＤ ＮＯ：２０）及び逆方向（５’−ＧＧＧＴＧＧＴＣＣＣＧＣＴＡＧＣＴＡＴＴＡＧＴＡＧＧＧＡＣＣＧＧＧＧ−３’）（ＳＥＱ ＩＤ ＮＯ：２１）のプライマーのセットによるＰＣＲによってｍｓｅＩＲ遺伝子に対するオープンリーディングフレーム（ＯＲＦ）を増幅した。下線を引いた塩基は正方向プライマーのＮｄｅＩ切断部位の位置を示す。ベント（登録商標名）ＤＮＡポリメラーゼ、１ｘサーモポル緩衝液、１００μ１のＰＣＲ反応物中の鋳型としての５００ｎｇのＭｉｃｒｏｃｏｃｃｕｓ種（ＮＥＢ＃４４６）の染色体ＤＮＡ及びプライマーを用いてＰＣＲを行った。９５℃にて１５秒間、６８℃にて６０秒間、７２℃にて４５秒間から成るサイクルを２５回行った。キアクイックＰＣＲ精製プロトコールを用いて、得られた７００塩基対のＰＣＲ産物を精製し、クレノウ酵素で処理し、ＮｄｅＩで消化して、キアクイックＰＣＲ精製プロトコールを用いて、もう１回精製した。
【００９４】
ＭｓｅＩ制限エンドヌクレアーゼ遺伝子を含有する、得られた７００塩基対のＮｄｅＩ−平滑末端断片を、ＮｄｅＩとＳｔｕＩで消化したｐＶＲ−２４ベクターに連結し、 予めＭｓｅＩメチラーゼ遺伝子コンストラクト、ｐＮＫＲ１７０７ＭｓｅＩｍ−９で修飾した大腸菌ＥＲ２５０２細胞を、連結物で形質転換した。分析した１８の形質転換体のうち、３個がｍｓｅＩＲ遺伝子を含有していた。ＭｓｅＩ制限エンドヌクレアーゼ遺伝子を含有するＤＮＡ挿入体の配列を決定した後、ＭｓｅＩ制限エンドヌクレアーゼを製造するために、１個の組換えプラスミド、ｐＶＲ−２５を選択した。
【００９５】
（２）株の構築
宿主においてＬａｃＩリプレッサのコピー数を増やすために、米国特許出願 出願番号第０９／６８９，３５９号に記載されたように、株ＥＲ２８３３（Ｔ７ｌａｃＩｑ株）を構築した。
【００９６】
（３）別の宿主及び異なったレベルでＭｓｅＩメチラーゼを発現するプラスミドと組合せた大腸菌におけるＭｓｅＩ制限エンドヌクレアーゼの過剰発現の最適化大腸菌におけるＭｓｅＩ制限エンドヌクレアーゼの過剰発現を最適化するために、これらＭｓｅＩメチラーゼを発現するプラスミド（ｐＮＫＲ１７０７ＭｓｅＩｍ−９、ｐＣＲ−２６及びｐＶＲ−２７）の１つを持つことによってＭｓｅＩエンドヌクレアーゼの自己消化から事前に保護された、発現株、ＥＲ２５６６／ｐＣＥＦ−８にｐＶＲ−２５を移入した。ＥＲ２５６６／ｐＣＥＦ−８は、誘導可能なｌａｃプロモータ及び、低レベルのＴ７リゾチームとＴ７ＲＮＡポリメラーゼの天然阻害を特徴とするｐＳＹＸ２０を基にしたプラスミド、ｐＣＥＦ−８の制御のもと、Ｔ７ＲＮＡポリメラーゼに対する遺伝子の染色体コピーを含有する宿主株である。さらなる情報については、Ｍｏｆｆａｔｔ，Ｂ．Ａ．及びＳｔｕｄｉｅｒ，Ｆ．Ｗ．の『Ｔ７リゾチームはＴ７ＲＮＡポリメラーゼによって転写を阻害する』（Cell, 49:221-227, 1987）を参照のこと。誘導されない細胞では、リゾチームはＴ７ＲＮＡポリメラーゼの基礎的な活性を低下させ、発現宿主中で安定に維持することができる標的遺伝子の範囲を増やす。さらに、Ｆ’エピゾーム上にｌａｃＩｑ遺伝子を１コピー有するもう１つの発現株、ＥＲ２８３３／ｐＣＥＦ−８を用いた。
【００９７】
全体として、大腸菌におけるＭｓｅＩ制限エンドヌクレアーゼ発現の研究には６株を用いた（表２）。株はすべて、ＭｓｅＩ制限エンドヌクレアーゼを発現するｐＶＲ−２５プラスミド及びＴ７バクテリオファージのリゾチーム遺伝子をコードするｐＣＥＦ−８プラスミドを含有する。ＭｓｅＩ制限エンドヌクレアーゼの高い収量について選択するために、種々の増殖条件を用いて、形質転換された宿主細胞を増殖させた。最適化実験で好ましい培地は、ルリア−ベルターニ（１リットル当り１グラムのグルコースと１グラムのＭｇＣｌ_２を補った；以後補完ＬＢという）培地であった。
【００９８】
増殖条件は以下のとおりであった。
ＭｓｅＲＭ１： 個々のコロニーからの細胞を０．５リットルのＬＢ培地中で４２℃にて８時間増殖させ、その後、０．２ｍＭの最終濃度でＩＰＴＧを加えてＴ７ＲＮＡポリメラーゼを誘導し、３０℃にて一晩（１５時間）細胞を増殖させた。必要に応じて抗生物質：３０μｇ／ｍｌのカナマイシン、１００μｇ／ｍｌのアンピシリン及び３０μｇ／ｍｌのクロラムフェニコールを加えた。最後に遠心によって培養したものを回収し、−２０℃に凍結した。
【００９９】
ＭｓｅＲＭ３： 各実験について、個々のコロニーからの細胞を０．５リットルのＬＢ培地中で３０℃にて一晩（１７時間）増殖させ、その後、０．２ｍＭの最終濃度でＩＰＴＧを加えてＴ７ＲＮＡポリメラーゼを誘導し、細胞を４時間増殖させ続けた。必要に応じて抗生物質：３０μｇ／ｍｌのカナマイシン、１００μｇ／ｍｌのアンピシリン及び３０μｇ／ｍｌのクロラムフェニコールを加えた。最後に遠心によって培養したものを回収し、−２０℃に凍結した。
【０１００】
ＭｓｅＲＭ４、ＭｓｅＲＭ５及びＭｓｅＲＭ６： 細菌培養物は５０％グリセロール中、−７０℃における凍結保存溶液として保持された。播種に用いた培養物は、単一コロニーを得るために適当な抗生物質を含有したＬＢプレート上に筋状に播いた。個々のコロニーを１ｍｌのＬＢ培地に再浮遊し、３０μｇ／ｍｌのカナマイシン、１００μｇ／ｍｌのアンピシリン及び３０μｇ／ｍｌのクロラムフェニコールを補った５００ｍｌのＬＢ培地を含有する１０００−ｍｌのフラスコに播種した。振盪培養器で３７℃、２５０ｒｐｍにて細胞を一晩（１６時間）増殖させた。その後、最終濃度０．２ｍＭでＩＰＴＧを加えた。さらに４時間細胞を培養し、次いで４℃で５分間８，０００ｒｐｍにて遠心することによって細胞を回収し、−２０℃にて凍結した。
【０１０１】
高レベルの発現クローンを同定するために２つの好ましい制限エンドヌクレアーゼアッセイを用いた。
超音波破砕法： 誘導した細胞（５００ｍｌ）を回収し、１０ｍＭのトリスＨＣｌ（ｐＨ７．５）及び１ｍＭのＥＤＴＡを含有する超音波破砕用緩衝液２０ｍｌに再浮遊した。氷上にてマクロ−チップのプローブで３０秒間の衝撃を４回行うことによって細胞を超音波破砕した。粗抽出物の一部をＮＥＢ緩衝液２緩衝液（５０μｌ）中のラムダＤＮＡ（１μｌ）に加え、３７℃にて１時間インキュベートした。０．８％のゲル電気泳動によってＤＮＡを分画し、ＥｔＢｒ染色によって視覚化した。
【０１０２】
エクスプレス法： 一晩、培養物又は誘導培養物（１０〜５００ｍｌ）を１ｍｌ回収し、５０ｍＭのトリスＨＣｌ、ｐＨ７．５及び２５％（容量／容量）のスクロースを含有する緩衝液０．２ｍｌに再浮遊し、溶液が均質になるまで混合した。２００ｍＭのＥＤＴＡ（ｐＨ８．０）を１１μｌそれに０．２５ＭトリスＨＣｌ（ｐＨ８．０）中で新しく調製した１０ｍｇ／ｍｌのリゾチームを２００μｌ溶液に加え、氷上で５分間インキュベートした。次いで１１．５μｌの１Ｍ、ＭｇＣｌ_２及び２４．２μｌの５％（容量／容量）Ｂｒｉｊ−５８を加えた。溶液をゆっくり混合し、室温にて１５分間インキュベートした。インキュベートした後、４℃にて１５分間、微量遠心機の最大速度で細胞粗溶解物を遠心した。上清をピペットにより新しいエッペンドルフ管に移し、必要になるまで氷上で保管した。３７℃にて１時間、ＭｓｅＩ反応緩衝液（ＮＥＢ緩衝液２）中で細胞抽出物の連続希釈によって、ラムダＤＮＡ基質（１．０μｇ）を消化した。電気泳動によりＤＮＡを分画し、ＥｔＢｒ染色によって視覚化した。ラムダＤＮＡのＭｓｅＩ消化と関係する適当なサイズのバンドの存在によって活性を決定した。
【０１０３】
ＭｓｅＩ制限エンドヌクレアーゼ発現の最適化に関する結果を表３に要約した。
【０１０４】
ＭｓｅＲＭ１株によって様々な収量のＭｓｅＩ制限エンドヌクレアーゼ（０．０８〜０．５ｘ１０^６Ｕ／ｇ細胞の湿重量）が得られた。細胞は緩やかに増加し、遅延時間はことのほか長かった。
【０１０５】
ｌａｃを基にした組換え発現システムの安定性及び再現性を高めるために、Ｆ’エピゾーム上にｌａｃＩ_ｑ遺伝子を１コピー有する新しい宿主株ＥＲ２８３３を構築した（米国特許出願、出願番号第０９／６８９，３５９）。実際、ｌａｃＩ_ｑ宿主（ＭｓｅＲＭ３）における発現の安定性及びプラスミドの維持性は著しく高められた：ＭｓｅＩ制限エンドヌクレアーゼの収量は、０．５〜１．４ｘ１０^６Ｕ／ｇ細胞の湿重量であった。この株（実施例４を参照のこと）から精製されたＭｓｅＩ制限エンドヌクレアーゼは、実質的に非特異的エンドヌクレアーゼ及びエクソヌクレアーゼを含まず、最終的な収量は約１５０，０００Ｕ／ｇであった。それは、元来のＭｉｃｒｏｃｏｃｃｕｓ種（ＮＥＢ＃４４６）から得られるよりも約１００倍多かった。
【０１０６】
【表３】

【０１０７】
残念ながら、ＭｓｅＲＭ３株は、−７０℃で保存し、回復させた後、ＭｓｅＩ制限エンドヌクレアーゼ活性を示さなかった。この問題を解決するために、ｐＶＲ−２６及びｐＶＲ−２７（上記実施例４）を構築することによってＭｓｅＩメチラーゼの発現レベルを高めた。このような株（ＭｓｅＲＭ４、ＭｓｅＲＭ５及びＭｓｅＲＭ６）では、−７０℃で株を保存した後でさえ高いＭｓｅＩ制限エンドヌクレアーゼ収量が得られ、ＭｓｅＲＭ４（ＮＥＢ＃１２８４；米国マサチューセッツ州、ベバリー、ニューイングランド・バイオラボズ社）は、１００リットルの製造用発酵槽でのスケールアップに用いられた（実施例４を参照のこと）。この大規模発酵でのＭｓｅＩ制限エンドヌクレアーゼの収量は細胞の湿重量当り０．５ｘ１０^６Ｕ／ｇであった。
【０１０８】
実施例６：組換えＭｓｅＩ制限エンドヌクレアーゼの製造
１００リットルの発酵槽において、晩期対数増殖期に増殖した組換え大腸菌株ＮＥＢ＃１２８４からＭｓｅＩ制限酵素を製造した。この細胞の試料を、２０００年８月２８日、ブタペスト協定の規約と条件のもとにＡＴＣＣに供託し、「ＡＴＣＣ受入番号ＰＴＡ−２４２１」を受け取った。
【０１０９】
（Ａ）細胞の増殖
５０％グリセロール中−７０℃での凍結保存溶液として、組換えＭｓｅＩ制限エンドヌクレアーゼを含有する形質転換された大腸菌宿主、ＮＥＢ＃１２８４を保存した。播種用に用いた培養物を、アンピシリン（１００μｇ／ｍｌ）、クロラムフェニコール（３０μｇ／ｍｌ）及びカナマイシン（５０μｇ／ｍｌ）を含有するＬＢ寒天プレートに筋状に播き、３７℃にて一晩インキュベートして単一コロニーを得た。３０μｇ／ｍｌのカナマイシン、１００μｇ／ｍｌのアンピシリン及び３０μｇ／ｍｌのクロラムフェニコールを補った１０ｍｌのＬＢ培地に播種するのに数個のコロニーを用いた。３７℃、２５０ｒｐｍの振盪培養器にて細胞を３時間増殖させ、次いで３０℃にてさらに３．５時間増殖させた（培養物の過剰増殖を回避するため）。この培養の最終的に補正されたクレットは１２２又は対数増殖期の中ほどであった。この培養物を用いて、３０μｇ／ｍｌのカナマイシン、１００μｇ／ｍｌのアンピシリン及び３０μｇ／ｍｌのクロラムフェニコールを補った１００リットルのＬＢに播種した。２ＳＣＦＭの通気（１分当りの標準立方フィート）及び２００ｒｐｍの撹拌速度で３０℃にて１８時間、発酵を行った。最終補正クレットは３１３であった。この発酵から３３１グラムの細胞（湿重量）を連続流れ方式の遠心によって回収し、−７０℃に保存した。１ｇの細胞から粗抽出物を作成し、上記の方法（実施例５を参照のこと）を用いて、酵素活性を推定した。 粗抽出物中のＭｓｅＩ制限エンドヌクレアーゼの収量は５００，０００＃Ｕ／ｇであり、それは、Ｍｉｃｒｏｃｏｃｃｕｓ種（ＮＥＢ＃４４６）の粗抽出物に比べて約１００倍高い。
【０１１０】
（Ｂ）ＮＥＢ＃１２８４からのＭｓｅＩ制限エンドヌクレアーゼの精製
以下の工程はすべて氷上にて又は４℃のどちらかにて行った。９９０ｍｌの緩衝液Ａ（０．１５ＭのＮａＣｌ、１０ｍＭのトリスＨＣｌ、ｐＨ７．５、１０ｍＭのＢＭＥ、１ｍＭのＥＤＴＡ及び５％（容量／容量）のグリセロール）に３３０グラムの細胞を浮遊し、０．５６のＯ．Ｄ．までガウリン・プレスによってｐｓｉｇ１２Ｋにて４回破砕した。ＰＥＧ６０００を７．５％でＮａＣｌを０．５Ｍで加えることにより、１１５０ｍｌの上清をＰＥＧ沈殿し、次いで４℃にて５０分間インキュベートした。４℃にて１２Ｋで３０分間、ＰＥＧスラリーを遠心した。５８０ｍｌの上清をＮａＣｌを含まない緩衝液Ａで０．１ＭのＮａＣｌに希釈し、緩衝液Ａで平衡化した４３０ｍｌのヘパリンハイパーＤカラムにかけた。１２００ｍｌの緩衝液Ａでカラムを洗浄し、次いで４０００ｍｌの０．１ＭのＮａＣｌ〜１．０ＭのＮａＣｌの直線勾配を適用した。０．２５Ｍ〜０．３５ＭのＮａＣｌで制限酵素活性が溶出し、それをプールした。ヘパリンハイパーＤプールをＮａＣｌを含まない緩衝液Ａで０．１ＭのＮａＣｌに希釈し、緩衝液Ａで平衡化した８８ｍｌのＰＥＩカラムにかけた。１００ｍｌの緩衝液Ａでカラムを洗浄し、１０００ｍｌの０．１Ｍ〜１．７ＭのＮａＣｌの直線勾配を適用した。０．７〜０．９ＭのＮａＣｌで制限酵素活性が溶出し、緩衝液Ｃ（５０ｍＭのＮａＣｌ、１５ｍＭのトリスｐＨ７．５、１０ｍＭのＢＭＥ、０．１ｍＭのＥＤＴＡ及び５％（容量／容量）のグリセロール）に対して一晩透析し、緩衝液Ｃで平衡化した２０ｍｌのソースＱカラムにかけた。４０ｍｌの緩衝液Ｃでカラムを洗浄し、４００ｍｌの０．０５ＭのＮａＣｌ〜１．０ＭのＮａＣｌの直線勾配を適用した。０．２５Ｍ〜０．３５ＭのＮａＣｌで制限酵素活性が溶出し、それをプールした。ソースＱプールを緩衝液Ｄ（１０ｍＭのＫＰＯ４、ｐＨ７．０、０．０７５ＭのＮａＣｌ、１０ｍＭのＢＭＥ、０．１ｍＭのＥＤＴＡ、及び５％（容量／容量）のグリセロール）に対して透析し、緩衝液Ｄで平衡化した２０ｍｌのヘパリンＴＳＫカラムにかけた。４０ｍｌの緩衝液Ｄでカラムを洗浄し、４００ｍｌの緩衝液Ｄ中の０．０７５Ｍ〜１．０ＭのＮａＣｌの直線勾配を適用した。０．３〜０．４ＭのＮａＣｌで制限酵素活性が溶出し、それをプールした。最終濃度１００μｇ／ｍｌでＢＳＡを加えた。プールしたものを保存用緩衝液（２０ｍＭのトリスＨＣｌ、ｐＨ７．５、０．１ＭのＥＤＴＡ、１ｍＭのＤＴＴ、５０ｍＭのＮａＣｌ、５０％（容量／容量）のグリセロール、２００μｇ／ｍｌのＢＳＡ）に対して一晩透析した。この精製法で２６，０００，０００単位のＭｓｅＩ制限エンドヌクレアーゼが得られた。この精製で得られたＭｓｅＩ制限エンドヌクレアーゼは実質的に非特異的なエンドヌクレアーゼ及びエクソヌクレアーゼを含んでいなかった。
【０１１１】
以下の基準を見ることによってＭｓｅＩ制限エンドヌクレアーゼ調製物の純度をチェックした。
【０１１２】
１．連結：ラムダＤＮＡの５倍過剰消化の後、生じたＤＮＡ断片の９５％を越えるものが、Ｔ４ＤＮＡリガーゼと連結された（１６℃にて１〜２μＭの５’末端濃度）。この連結断片のうち９５％が再切断可能であった。
【０１１３】
２．持続消化：１μｇのラムダと１００単位の酵素を含有する５０μｌの反応物を１６時間インキュベートした後、１単位の酵素で１時間行われた反応としてＤＮＡバンドの同じバンドパターンが生じた。
【０１１４】
３．エクソヌクレアーゼ活性： １μｇの超音波破砕した３Ｈ ＤＮＡ（１０５ｃｐｍ／μｇ）を含有する５０μｌの反応において３７℃にて４時間、１００単位の酵素をインキュベートした後、０．４％未満の放射活性が放出された。
【０１１５】
試験はすべて、以下の反応緩衝液：ＮＥＢ緩衝液２（５０ｍＭのＮａＣｌ、１０ｍＭのＭｇＣｌ_２、１０ｍＭのトリスＨＣｌ、１ｍＭのＤＴＴ、２５℃にてｐＨ７．９、１００μｇ／ｍｌのＢＳＡ補完）で行った。単位の決定：５０μｌの１ｘＭｓｅＩ反応緩衝液（ＮＥＢ緩衝液２）中で、３７℃にて１時間、連続希釈したＭｓｅＩエンドヌクレアーゼでラムダＤＮＡ基質（１．０μｇ）を消化した。電気泳動によってＤＮＡを分画し、ＥｔｄＢｒによって視覚化した。ラムダＤＮＡのＭｓｅＩ消化に関係する適当なサイズのバンドの存在によって活性を決定した。制限エンドヌクレアーゼ活性の１単位は、特定したＮＥＢ緩衝液を用いて、５０μｌの総反応容量中の１μｇの基質ＤＮＡを１時間で完全に消化するのに必要な量として定義される。
【０１１６】
【配列表】

【図面の簡単な説明】
【図１】ＭｓｅＩＤＮＡメチルトランスフェラーゼ遺伝子をコードする組換えプラスミドｐＶＲ１８の制限地図を示す。
【図２】推定上のＤＮＡメチルトランスフェラーゼ遺伝子をコードする組換えプラスミドｐＥｓａＤｉｘ４Ｉの制限地図を示す。
【図３】推定上のＤＮＡメチルトランスフェラーゼ遺伝子をコードする組換えプラスミドｐＥｓａＤｉｘ５Ｉの制限地図を示す。
【図４】推定上のＤＮＡメチルトランスフェラーゼ遺伝子をコードするｐＥｓａＤｉｘ４ＩプラスミドのＭｓｅＩに対する感受性の寒天ゲル解析を示す。レーン１、切断されていないｐＥｓａＤｉｘ４Ｉ；レーン２、１０単位のＭｓｅＩと共に一晩インキュベートした後のｐＥｓａＤｉｘ４Ｉ；レーン３、切断されていないｐＵＣ１９；レーン４、１０単位のＭｓｅＩと共に一晩インキュベートした後のｐＵＣ１９；レーン４、１０単位のＭｓｅＩと共に一晩インキュベートした後のｐＥｓａＤｉｘ４Ｉ＋ｐＵＣ１９；レーン５、分子量の基準（１ｋｂのＤＮＡラダー、ニューイングランドバイオラボズ社）。
【図５】推定上のＤＮＡメチルトランスフェラーゼ遺伝子をコードするｐＥｓａＤｉｘ５ＩプラスミドのＭｓｅＩに対する感受性の寒天ゲル解析を示す。レーン１、切断されていないｐＥｓａＤｉｘ５Ｉ；レーン２、１０単位のＭｓｅＩと共に一晩インキュベートした後のｐＥｓａＤｉｘ５Ｉ；レーン３、切断されていないｐＵＣ１９；レーン４、１０単位のＭｓｅＩと共に一晩インキュベートした後のｐＵＣ１９；レーン４、１０単位のＭｓｅＩと共に一晩インキュベートした後のｐＥｓａＤｉｘ５Ｉ＋ｐＵＣ１９；レーン５、分子量の基準（１ｋｂのＤＮＡラダー、ニューイングランドバイオラボズ社）。
【図６】ｍｓｅＩＭ遺伝子のＤＮＡ配列（ＳＥＱ ＩＤ ＮＯ：１）及びそれにコードされたアミノ酸配列（ＳＥＱ ＩＤ ＮＯ：２）を示す。
【図７】ｅｓａＤｉｘ４ＩＭ遺伝子のＤＮＡ配列（ＳＥＱ ＩＤ ＮＯ：３）及びそれにコードされたアミノ酸配列（ＳＥＱ ＩＤ ＮＯ：４）を示す。
【図８】ｅｓａＤｉｘ５ＩＭ遺伝子のＤＮＡ配列（ＳＥＱ ＩＤ ＮＯ：５）及びそれにコードされたアミノ酸配列（ＳＥＱ ＩＤ ＮＯ：６）を示す。
【図９】ｍｓｅＩＲ遺伝子のＤＮＡ配列（ＳＥＱ ＩＤ ＮＯ：７）及びそれにコードされたアミノ酸配列（ＳＥＱ ＩＤ ＮＯ：８）を示す。
【図１０】エラー傾向ＰＣＲによって無作為に変異誘発された構成プロモータのライブラリの構築に用いられた組換えプラスミドｐＮＫＲ１７０７ｍｓｅＩＭの制限地図を示す。
【図１１】ＭｓｅＩ ＤＮＡメチルトランスフェラーゼ遺伝子及び上流の調節要素をコードする組換えプラスミドｐＮＫＲ１７０７ｍｓｅＩＭ−９の制限地図を示す。
【図１２】最適プロモータ配列を含有するＭｓｅＩ ＤＮＡメチルトランスフェラーゼ遺伝子（ＳＥＱ ＩＤ ＮＯ：９）の上流のＤＮＡ配列を示す。
【図１３】ＭｓｅＩ ＤＮＡメチルトランスフェラーゼ遺伝子の制御された発現に用いられたプラスミドｐＶＲ−２６及びｐＶＲ−２７の構築を示す。
【図１４】ｐＶＲ−２４発現ベクターの構築を示す。
【図１５】ＭｓｅＩ制限エンドヌクレアーゼ遺伝子をコードするｐＶＲ−２５の制限地図を示す。
【図１６】Ｔ７リゾチーム遺伝子をコードするｐＣＥＦ−８の制限地図を示す。
【図１７】細胞毒性タンパク質をコードする遺伝子をクローニングするためのｐＶＲ−２５における厳密な調節システムの作用機序を示す。
【図１８】大腸菌ＭｓｅＲＭ４、ＭｓｅＲＭ５及びＭｓｅＲＭ６から作製された細胞粗抽出物におけるＭｓｅＩ制限エンドヌクレアーゼ活性のアッセイを示す。増殖条件は実施例６に記載されている。
【図１９】１００リットル発酵槽における増殖後の、大腸菌株ＭｓｅＲＭ４（ＮＥＢ＃１２８４）から作製された細胞粗抽出物でのＭｓｅＩ制限エンドヌクレアーゼ活性のアッセイを示す。[0001]
BACKGROUND OF THE INVENTION
The present invention, in one embodiment, so that expression corrects changes in the physiological state of the cells and thus preferably provides complete protection against cleavage by a cognate restriction endonuclease at any growth stage, Cloning the restriction modification system of interest, including first carrying out the method to produce a balanced level of protectively modified methyltransferase activity; then introducing a restriction endonuclease and providing its expression. Relates to a method of expression.
[0002]
[Prior art]
Restriction endonucleases belong to a class of enzymes called nucleases that degrade or cleave single- or double-stranded DNA. Restriction endonucleases act by recognizing and binding specific nucleotide sequences (“recognition sequences”) along a DNA molecule. Once bound, the endonuclease cleaves the molecule within or on the recognition sequence. Although the position is fixed for any arbitrary endonuclease, the position of cleavage may differ between the various restriction endonucleases. Different restriction endonucleases have different affinities for recognition sequences. Among the thousands of bacterial and archaeal species examined to date, over 200 restriction endonucleases have been identified that recognize unique specificity.
[0003]
Restriction endonucleases are classified based on their composition and the need for cofactors, the nature of the target sequence, and the location of the DNA cleavage site relative to the target sequence (Yuan, R., Ann. Rev. Biochem., 50: 285 -315, 1981). Currently, three well-characterized restriction endonucleases are known (I, II and III). Type I enzymes recognize specific sequences but cleave randomly with respect to that sequence. Type III restriction endonucleases recognize specific sequences and cleave at defined positions relative to one of the sequences, but never digest completely. Neither of these two enzymes is suitable for practical use. Type II restriction endonucleases recognize specific sequences (4-8 nucleotides in length) and cleave at defined positions within or very close to that sequence. Usually they are Mg ²⁺ Requires only ions. If type II restriction endonucleases are purified from other bacterial components, they can be used in the laboratory to cleave DNA molecules into specific fragments. This property allows researchers to manipulate the DNA molecule and analyze the resulting structure.
[0004]
Bacteria, at best, tend to process only a few restriction endonucleases. Restriction endonucleases are named by three letter acronyms derived from the name of the organism in which they occur (Smith and Nathans, J. Mol. Biol., 81: 419-423, 1973). The first letter is from the genus and the second and third letters are from the species. Thus, for example, a strain of Deinococcus radiophilus species synthesizes three different type II restriction endonucleases, designated DraI, DraII and DraIII. Each such enzyme recognizes and cleaves the sequences, TTTAAA, PuGGNCCCPy and CACNNNNGTG. On the other hand, the RY13 strain of Escherichia coli synthesized only one type II restriction enzyme, EcoRI, which recognizes the sequence GAATTC (Roberts RJ, and Macelis D., Nucl. Acids Res., 28: 306-7 , 2000).
[0005]
The second component of the bacterial and archaeal restriction system is a modified methylase (Roberts and Halford, in 'Nucleases' 2nd ed., Linn et al., Ed.'S, p. 35-88, 1993). Such enzymes are complementary to restriction endonucleases and provide a means by which bacteria can protect their own DNA and be distinguished from foreign, invading DNA. A modified methylase recognizes and binds to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving DNA, chemically adds one or other nucleotides within the sequence by adding a methyl group. Qualify. After methylation, the recognition sequence is no longer cleaved by the restriction endonuclease. By virtue of the activity of the modified methylase, the DNA of the bacterial cell is modified and is therefore not sensitive to the presence of endogenous restriction endonucleases. Only non-modified, and thus distinguishable, foreign DNA is susceptible to restriction endonuclease recognition and cleavage.
[0006]
In nature, type II restriction endonucleases are believed to cleave foreign DNA, such as virus and plasmid DNA, if the DNA has not been modified by appropriate modifying enzymes (Wilson and Murray, Annu. Rev. Genet., 25: 585-627, 1991). In this way, the cells are protected from invasion by foreign DNA. Thus, it is widely believed that the evolution of type II restriction modification systems has been driven by the need for cells to protect themselves from infection by foreign DNA (cellular defense hypothesis).
[0007]
With the advent of genetic engineering techniques, it is now possible to clone genes and produce proteins and enzymes encoded by them in larger quantities than those available with conventional purification techniques. The key to isolating clones of restriction endonuclease genes is to develop a simple and reliable method for identifying such clones in gene libraries. One possible difficulty is the restriction end because the transcription and translation machinery of the source organism is different from that of E. coli, such as differences in promoter or ribosome binding sites or gene codon composition. Some nuclease genes and methylase genes may not be expressed in E. coli. Isolation of a methylase gene requires that the methylase be fully expressed in E. coli and fully protect at least a portion of the plasmid carrying the gene. Isolation of the active form of endonuclease requires that the methylase be fully expressed to fully protect the host DNA or to prevent lethal damage resulting from endonuclease cleavage. Another obstacle to cloning restriction endonucleases is that some E. coli strains react detrimentally to cytosine or adenine modifications; methylated cytosines (Raleigh and Wilson, Proc. Natl. Acad. Sci., USA 83: 9070- 9074, 1986) or methylated adenine (Heitman and Model, J. Bact., 196: 3243-3250; Raleigh et al., Genetics, 122: 279-296, 1989; Waite-Rees, et al., J. Bacteriology , 173: 5207-5219, 1991). Cytosine-specific or adenine-specific methylase genes cannot be easily cloned in such strains, either with themselves or their corresponding endonuclease genes. To circumvent this problem, a mutant strain of E. coli lacking such a system (McrA ⁻ And McrB ⁻ Or Mrr ⁻ ) Is required.
[0008]
Several approaches have been used to clone restriction genes into E. coli:
(1) Selection based on phage restriction
In the first cloned system, bacteriophage infection was used as a means to identify and select restriction endonuclease clones (EcoRII: Kosykh et al., Molec. Gen. Genet., 178: 717-719, 1980). HhaII: Mann et al., Gene 3: 97-112, 1978; PstI: Walder et al., Proc. Nat. Acad. Sci., USA 78: 1503-1507, 1981). The presence of restriction-modification systems in bacteria allows the bacteria to resist bacteriophage infection, so cells carrying cloned restriction-modification genes are in principle selected as survivors from libraries exposed to phage. Isolated. However, this method has been found to have only limited advantages. Specifically, it has been found that cloned restriction-modification genes do not always exhibit sufficient phage resistance to provide selective survival under normal conditions.
[0009]
(2) Selection based on vector modification
A second approach used to clone a growing number of systems involves the selection of active methylase genes (US Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acid Res., 13: 6403-6421, 1985). Because restriction genes and modification genes are often closely linked, it is often possible to clone both genes simultaneously. This selection does not always result in a complete restriction system, but may instead only obtain the methyltransferase gene (BspRI: Szomolanyi et al., Gene 10: 219-225, 1980; BcnI: Janulaitis et al., Gene 20: 197-204, 1982; BsuRI: Kiss and Baldauf, Gene 21: 111-119, 1983; and MspI: Walder et al., J. Biol. Chem., 258: 1235-1241, 1983. ).
[0010]
(3) Subcloning of natural plasmid
Another cloning method is a transfer system originally characterized as a plasmid for cloning into plasmid-derived E. coli (EcoRVI: Bougueleret et al., Nucl. Acid Res., 12: 3659-3676, 1984; PaeR7I : Gingeras and Brooks, Proc. Natl. Acad. Sci., USA 80: 402-406, 1983; Theriault and Roy, Gene 19: 355-359, 1982; PvuII: Blumenthal et al., J. Bacteriol., 164: 501-509, 1985).
[0011]
(4) Multiple-step cloning
Direct methylase selection methods may fail to obtain methylase (and / or endonuclease) clones due to various obstacles. See, for example, pages 25-32 of Lunnen et al., Gene 74 (1) (1988). One potential obstacle to cloning restriction-modification genes is to attempt to introduce the endonuclease gene into a host that has not been previously protected by modification. When the methylase gene and endonuclease gene are introduced together as a single clone, the methylase must protectively modify the host DNA before the endonuclease has the opportunity to cleave. Thus, sometimes it may be possible to clone genes first, then methylase and then endonuclease (see US Pat. No. 5,320,957).
[0012]
(5) Selection based on induction of SOS reaction that can be induced by DNA damage
Another method for cloning methylase and endonuclease genes is based on a colorimetric assay for DNA damage (see US Pat. No. 5,492,823). When screening for methylase, a sensitive host E. coli strain such as AP1-200 is transformed with the plasmid library. By expression of methylase, McrA ⁺ , McrBC ⁺ Or Mrr ⁺ SOS reaction is induced in E. coli strains. The AP1-200 strain contains lacZ fused to the dinD locus that is temperature sensitive to the Mcr and Mrr systems and is inducible by E. coli damage. Detection of recombinant plasmids encoding methylase or endonuclease is based on induction of lacZ at a limited temperature. Transformants encoding the methylase gene are detected as blue colonies on LB agar plates containing X-gal (Piekarowicz et al., Nucleic Acids Res., 19: 1831-1835, 1991; and Piekarowicz et al. , J. Bacteriology 173: 150-155, 1991). Similarly, the E. coli strain, ER1992, contains a dinD1-lacZ fusion but lacks a methylation-dependent restriction system, McrA, McrBC and Mrr. This system (called the “endo-blue” method) detects endonuclease genes in the absence of cognate methylases when endonucleases damage host cell DNA, including SOS reactions. can do. On LB agar plates supplemented with X-gal, SOS-induced cells form deep blue colonies (Fomenkov et al., Nucleic Acids Res., 22: 2399-2403, 1994, and US Pat. No. 5). , 498, 535).
[0013]
(6) Synonymous inverse PCR based on N-terminal sequence
The modified methyltransferase gene cannot be identified (see US Pat. No. 5,945,288) or the methylase gene can be identified but the open reading frame specifying the restriction endonuclease is not clear Things can happen. In these cases, additional methods for identifying endonuclease genes can be particularly applied when the restriction endonuclease can be purified from the source organism in sufficient quantity and purity. In this method, the endonuclease is purified to substantial homogeneity and the polypeptide is sequenced. The resulting polypeptide sequence can be back-translated into a DNA sequence and synonymous PCR primers designed to amplify a portion of the endonuclease gene from the genomic DNA of the source organism or from a gene library created therefrom. it can. The complete gene DNA sequence can be obtained by Southern blot analysis or by further direct or inverse PCR methods. If a cognate methyltransferase is not obtained or cannot be expressed, the stability and convenience of a restriction endonuclease clone alone is usually severely compromised.
[0014]
Although the above methods may allow the production of specific systems of methyltransferases and restriction endonucleases, the establishment of strains that can still be used for enzyme production is problematic. The difficulty is often with the expression of methyltransferase at a suitable level. This is particularly true with method (6). Stabilize such clones with heterospecific methyltransferases that are not related to the endonuclease in the original host but recognize the same or related sequences and prevent the endonuclease from cleaving the recognition sequence (See US Pat. No. 6,048,731).
[0015]
It can happen that there is no suitable heterospecific methyltransferase available and that the degree of protection afforded to the host by the cognate methyltransferase is inadequate; or clearly obtaining an appropriate level of methyltransferase Although it can occur that such levels are toxic to the cells and result in strains that cannot be stored; or protection is clearly adequate and the protected cells are viable Possible, but may result in a strain that does not express detectable endonuclease by the combination of methyltransferase and endonuclease gene; or protection is clearly appropriate and the combination of methyltransferase and endonuclease gene Detectable by Producing strain expressing endonuclease, but commercially in the production of useful levels of enzyme is likely to be happen not sufficiently suitable.
[0016]
Numerous factors can be postulated that alter the required level of enzyme required to effectively protect the host cell from restriction endonuclease cleavage. Such factors require rapid growth in which there are larger amounts of DNA copies than are present in the cells during the stationary phase of growth; new synthesis of modified methyltransferases is required before new synthesis of restriction endonucleases begins. Recovery from possible dormancy; various starvation, for example, where the level of a required DNA methyltransferase cofactor such as S-adenosylmethionine may change; and DNA damage or other physiology Specific physiological conditions such as physical disturbances. Furthermore, the level of methyltransferase becomes too high, for example by binding to or methylating irrelevant sites related to the cognate site, making it toxic and thus reading the DNA sequence by regulatory or DNA condensed proteins. May interfere. Thus, in order to perpetuate indefinitely, the absolute level of methyltransferase expression may need to be varied depending on conditions over the duration of the culture.
[0017]
This need for a fine level of control is not unique to modified methyltransferases. Within 50 years of research, for example, it degrades nutrients or synthesizes essential components (trp, his), or reacts to harmful factors (DNA damage response, heat shock response), a set of catabolic and anabolic genes (lac A number of detailed adjustment schemes have been described for various functions such as ara, gal). Such regulatory effects are mediated, for example, by changes in promoter activity (due to activator or repressor), transcription stability (due to retroregulatory elements), translational level (weakening or translational linkage). Despite this high level of understanding, it is not easy to predict in advance how the demand for function will change with physiological changes and how to achieve the desired level of function.
[0018]
Because purified restriction endonucleases and, to a lesser extent, modified methylases are useful tools for characterizing genes in the laboratory, bacterial strains that synthesize such enzymes in large quantities via recombinant DNA technology can be used. There is a commercial motivation to obtain. Such strains are useful because they provide a means of manufacturing in commercially useful quantities, as well as simplify the purification operation.
[0019]
[Problems to be solved by the invention]
The present invention, in one embodiment, so that expression corrects changes in the physiological state of the cells and thus preferably provides complete protection against cleavage by a cognate restriction endonuclease at any growth stage, Cloning the restriction modification system of interest, including first carrying out the method to produce a balanced level of protectively modified methyltransferase activity; then introducing a restriction endonuclease and providing its expression. Relates to a method of expression.
[0020]
The invention further examines specifically the degree of protection during a critical growth phase, which may be selected from stationary phase, logarithmic growth phase, regeneration phase from storage or other growth phase, and then The present invention relates to a method for producing a balanced level of protectively modified methyltransferase activity comprising identifying suitable expression vectors by selecting for function in a critical growth phase.
[0021]
The above method is exemplified by the cloning and expression of the MseI restriction modification system, which is encoded by a DNA (deoxyribonucleic acid) fragment that contains two related enzymes, namely the DNA sequence 5′-TTAA-3 ′. And the enzyme that cleaves the phosphate diester bond between the T residues of this recognition sequence to produce a 5 'extension of 2 bases (Morgan RD, Nucl. Acids Res., 16: 3104, 1988) , MseI restriction endonuclease) and the same DNA sequence, 5′-TTAA-3 ′, but modified by adding a methyl group to prevent cleavage by MseI endonuclease, It encodes a second enzyme known as MseI. Furthermore, the invention is disclosed in M.C. It differs in sequence from MseI and It relates to two additional DNA fragments that perform the same function as MseI, ie, encode an enzyme that modifies the sequence 5′-TTAA-3 ′ by adding a methyl group to prevent cleavage by MseI endonuclease. The present invention also relates to a DNA fragment, a vector containing the DNA fragment, a method for preparing a transformed host containing the DNA fragment, and an improved method for producing an MseI restriction endonuclease from such a transformed host.
[0022]
[Means for solving the problems]
The MseI restriction endonuclease produced in accordance with the present invention is substantially pure and free of contaminants commonly found in restriction endonuclease preparations made by conventional techniques.
[0023]
The MseI methylase gene, but not the MseI endonuclease gene, was obtained according to techniques commonly cited for methylase selection (US Pat. No. 5,200,333, the disclosure of which is incorporated herein by reference). However, none of the clones obtained by methylase selection expressed detectable MseI restriction endonuclease activity and were not completely protected from MseI digestion after overnight incubation. Methylase clones were sequenced and the MseI methylase gene was identified based on homology with other N6-adenine methylases. Although the methylase clone did not produce detectable MseI endonuclease activity, it was speculated that the endonuclease gene appears to be located adjacent to the methylase gene. Therefore, DNA adjacent to the MseI methylase gene from Micrococcus species was amplified by inverse PCR technique and sequenced.
[0024]
In order to locate and reliably identify the MseI endonuclease gene, the N-terminal amino acid sequence of the highly purified MseI restriction endonuclease protein obtained from Micrococcus species was determined. An open reading frame in which the deduced amino acid sequence matched the N-terminal amino acid sequence of the MseI endonuclease was found in the DNA sequence obtained by the inverse PCR technique, and was located 3 ′ of the methylase gene. The MseI methylase gene was amplified and cloned with a vector compatible with a normal high expression vector. The MseI endonuclease gene was then amplified and ligated into an expression vector such as the pET series of vectors and introduced into a host previously modified with MseI methylase carried in another compatible vector; In such a construct, no MseI activity was found. The following further results indicate that this failure of MseI expression was due to inadequate expression of methylase, and successful endonuclease expression was a lethal event. After obtaining a fully modified vector according to the present invention, endonuclease expression was also carefully regulated by constructing a vector that suppressed endonuclease expression during cell growth prior to induction of the endonuclease gene. A host carrying the endonuclease gene and the methylase gene in such a special construct was then grown, induced, recovered and used to make the MseI endonuclease.
[0025]
A preferred method for cloning and expressing the MseI restriction-modification system consists of obtaining a methylase positive clone according to the methylase selection method and determining the DNA sequence of such an MseI methylase positive clone. DNA adjacent to the methylase gene is obtained by inverse PCR technique and sequenced. The MseI endonuclease protein from Micrococcus species is purified to near homogeneity and the N-terminal amino acid sequence is determined. The MseI endonuclease gene is identified based on DNA sequence and amino acid sequence data. The expression of MseI methylase is adjusted to achieve complete protection of the host genome without expressing so much methylase that it is toxic to the host. DNA obtained from cells in rapid logarithmic growth phase is examined for protection against cleavage of the MseI endonuclease and this complete methylation status is determined using a construct that provides complete protection under such rapid growth conditions. To monitor. The complete gene is then amplified from Micrococcus sp. Genomic DNA to limit expression of the MseI endonuclease during cell growth prior to induction such as pVR-24 (New England Biolabs, Beverly, Mass., USA) The MseI endonuclease is expressed by ligating to an expression vector designed as such. Expressed in another compatible plasmid with appropriate genetic composition that provides sufficient regulatory capacity (US patent application Ser. No. 09 / 689,359), providing complete protection against MseI cleavage. The construct is introduced into a host that has previously modified the MseI site by carrying the MseI methylase gene. The MseI endonuclease is produced by growing a host containing the MseI endonuclease gene and the methylase gene, including appropriate expression conditions, harvesting the cells, and then purifying the MseI endonuclease.
[0026]
DETAILED DESCRIPTION OF THE INVENTION
In one embodiment, the present invention protects DNA from cleavage by a restriction enzyme, preferably in such a manner that complete protection of the host DNA is observed at any growth stage where the cognate restriction endonuclease is present without becoming toxic. The present invention relates to a method for producing a target restriction endonuclease by first providing a vector that expresses a modified methyltransferase gene (a fully protecting methyltransferase vector) and then providing a vector that expresses a desired restriction endonuclease gene. The present invention is not limited by the properties of the modified methyltransferase or the restriction enzyme, except that the modified methyltransferase must protect against cleavage by a restriction enzyme.
[0027]
In a preferred embodiment, complete protection is provided by identifying regulatory elements capable of manipulating methyltransferase expression to provide complete protection during the growth phase that is particularly sensitive to methyltransferase expression patterns. A methyltransferase vector may be obtained. In accordance with the present invention, this may be performed by the following steps:
(1) obtaining a methyltransferase gene in a vector by a known method;
(2) placing a regulatory element such as a promoter in a suitable position with respect to the gene;
(3) transformation in a desired host cell;
(4) re-isolating the vector from the pooled transformants during the exponential growth phase;
(5) selection by digestion with endonuclease; and
(6) Retransformation of new hosts with surviving, undigested and thus protected vector populations.
[0028]
Step (4) of this method is from the exponential growth phase, or from various other growth phases, as appropriate, for example from the stationary phase, from the resting phase activated by starvation of carbon or nitrogen or other essential nutrients, or Even when carried out by pooled vectors isolated from cells in a special physiological state, such as a damaged state of DNA, or from cells in the presence of a cause of physiological injury, such as acidic media or toxic compounds Those skilled in the art will appreciate that it is good.
[0029]
In a preferred embodiment, the regulatory element of step (2) is identified by a method comprising the following steps. That is,
(A) cloning a pool of fragments containing a variety of different regulatory elements at a desired location into a vector containing a methyltransferase gene; and
(B) Continue the steps (3) to (6).
[0030]
It will be further appreciated by those skilled in the art that the selection method including steps (3) to (6) may be repeated to select further improvements.
[0031]
It will be further appreciated by those skilled in the art that step (2a) may be repeated, cloning the pool of fragments containing regulatory elements at the same or different positions, followed by selection as appropriate. .
[0032]
The present invention is not limited by the individuality of the regulatory element which may be a promoter, operator, enhancer or downstream regulatory element.
[0033]
In a preferred embodiment, the methyltransferase in step (1) is isolated by a methylase selection method (US Pat. No. 5,200,333). The present invention is not limited to the methyltransferase gene isolated by this method, but the above method, for example, identification based on phage selection, natural plasmid subcloning, induction of SOS reaction inducible by DNA damage, purified protein Includes genes identified in sequence databases from inverse PCR based on the amino acid sequence of, or similar to the sequence of other methyltransferases, followed by cloning by PCR or Southern blot based on methods (Kong, et al., Nucleic Acids Res., 28: 3216-3223, 2000).
[0034]
In a preferred embodiment, the collection of different regulatory elements in step (2a) is randomly performed by error-prone PCR along with contaminating chromosomal fragments that may be present in the mutagenized fragment preparation. Mutagenized S. cerevisiae Contains a copy of the tyhimurium his promoter. The present invention is not limited to the fragment thus obtained, but is a fragment isolated from genomic DNA of E. coli or another organism, or oligonucleotide synthesis by a random position or a synonymous sequence at a specific position Or collection of fragments generated by random or specific fragment collection recombinant PCR. In a preferred embodiment, the regulatory element thus obtained is the sequence SEQ ID NO: 9.
[0035]
The method according to the present invention may be applied to any methyltransferase that confers protection from cleavage by the relevant restriction endonuclease, not merely which co-occurs with the endonuclease in a particular natural isolate. Will be further understood by those skilled in the art.
[0036]
The present invention further relates to isolating a methyltransferase of the desired specificity from environmental resource DNA rather than first culturing the organism in which it is contained. In a preferred embodiment, such genes are methylase selected from DNA from prokaryotic mixed green filaments and pedestal colonies growing at 68 ° C. in Dixie Valley Hot Spring, Nevada, USA. Isolated by
[0037]
The present invention further relates to the supply of the desired restriction endonuclease gene expressed from the vector with strict regulation so that very low levels of protein are expressed in the absence of induction (accepting very low basic expression). ). In a preferred embodiment, the stringent regulatory vector is an antagonist that reads through a cloned gene of interest as described in WO 99/11821 and US patent application Ser. No. 09 / 486,356. Basic expression by providing a lower copy number than that present in the pre-existing vector pLT7K, which is used for this purpose, but contains vectors with a specific and independently regulatable promoter. It is even lower. In a preferred embodiment, the vector copy number is reduced by exchanging the origin of replication of pLT7K with that of pACYC184. Other origins of replication may also be used; for example, pSC101 (Stoker, et al., Gene 18: 335-341, 1982), pSYX20 (US Pat. No. 5,262,313), F (Shizuya, et al., Proc. Natl. Acad. Sci. USA 89 (18): 8794-8797. 1992) or other low copy number vectors (Harayama, et al., Mol. Gen. Genet., 184: 52- 55, 1981 and Wohlfarth, et al., J. Gen. Microbiol., 134: 433-440, 1988). In a preferred embodiment, the vector is pVR-24.
[0038]
In a preferred embodiment, a strain that expresses a high level of expression of a negative regulator in a direction capable of translating the gene of interest, as described in the accompanying US patent application, Ser. No. 09 / 701,626. By further reducing the basic expression level, it is achieved.
[0039]
The above method is illustrative in another embodiment of the invention, namely cloning and expression of the MseI restriction-modification system.
[0040]
The present invention also provides novel DNA constructs and novel compositions comprising microbial strains that produce the MseI restriction endonuclease. The restriction endonuclease, MseI, of interest in the present invention recognizes the DNA sequence 5′-TTAA-3 ′ and cleaves the phosphodiester bond between the T residues of this recognition sequence to produce 2 bases. 5 'elongation occurs.
[0041]
To overexpress the MseI restriction endonuclease, additional steps are required beyond the known methylase selection method (US Pat. No. 5,200,333), which is particularly toxic to the host. Included is expression of MseI methyltransferase that does not produce large amounts of methyltransferase but is well balanced to fully protect the host genomic DNA from MseI digestion in vivo. The E. coli host is first modified with a vector containing the mseIM gene optimized for expression so that complete protection against MseI endonuclease is observed even during very rapid cell growth (exponential growth phase). The host is then transformed with a suitable vector containing the mseIR gene, such as pVR-25, and colonies containing both vectors are then selected on a suitable antibiotic plate. Individual transformants are grown and identified for MseI endonuclease producing constructs by assaying for MseI endonuclease activity (as in Example 5 below).
[0042]
The method described herein for cloning and expressing the MseI methylase gene and the MseI restriction endonuclease gene preferably in E. coli employs the following steps.
1) Cloning of DNA methyltransferase gene that protects against cleavage by MseI
A DNA-modified methylase recognizes and binds to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, one or other nucleotides can be chemically altered within the sequence by adding methyl groups. Modification is well known. After this methylation, the recognition sequence is no longer bound by the restriction endonuclease and is not cleaved. The DNA of bacterial cells is always fully modified by its modified methylase and is therefore completely insensitive to the presence of endogenous restriction endonucleases. In this situation, only non-modified and thus identifiable foreign DNA is susceptible to restriction endonuclease recognition and attack. The first step of the method is to identify a DNA methyltransferase gene that protects against cleavage by MseI. To accomplish this, a Micrococcus species DNA methylase (NEB446) can be cloned. Alternatively, as described in US Pat. No. 5,179,015, DNA can be protected from RM systems other than the MseI RM system to prevent digestion by MseI restriction enzymes. DNA methyltransferases that can be identified can be identified. In the present invention, three types of methylase DNA that can protect DNA from digestion by MseI restriction enzyme have been identified.
[0043]
First, total genomic DNA was purified from Micrococcus species (S.coccus) (NEB # 446). A random library of this DNA was constructed by partially digesting the DNA with the frequently-cutting endonuclease, Sau3AI, to generate fragments approximately 1-10 kilobases (kb) in length. Such a fragment was ligated to the vector pBR322, which had been previously cleaved with BamHI and dephosphorylated. The ligation reaction was transformed into chemically competent E. coli ER2502 cells. The transformants were pooled and the plasmid was a population that was purified to form a primary plasmid library. A portion of this purified plasmid was digested with MseI to destroy all plasmids that did not express the MseI methylase gene in vivo and therefore did not protect the plasmid DNA from digestion. E. coli ER2502 was transformed again with the digested plasmid pool and the intact plasmid expressing MseI methylase was recovered. Individual clones were picked and plasmid DNA was purified and exposed to cleavage with MseI endonuclease. The plasmid that was not cut by MseI contained the MseI methyltransferase gene.
[0044]
In a preferred embodiment, the methyltransferase gene protects against the restriction endonuclease MseI available from Micrococcus sp. (NEB # 446), SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: You may choose between a set of sequences that can encode the protein identified in 7. Such proteins may be encoded, for example, by the DNA sequences set forth in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 6.
[0045]
To search for alternative methyltransferase DNA that can protect DNA from cleavage by MseI endonuclease, construct a library of clones derived from DNA sources other than Micrococcus species with vectors containing one or more MseI restriction sites May be. The library of clones is then selected by one or more digestions with MseI to destroy unprotected clones, followed by transformation with a digested plasmid to recover the protected clones. Such libraries include DNA isolated from prokaryotic mixed green filaments and pedestal colonies growing at 68 ° C. in Dixie Valley Hot Spring, Nevada, USA (“DNA from the Environment”). Named). DNA from the purified environment was digested with NsiI endonuclease, ligated to the vector pNEB193 previously cut with PstI restriction endonuclease and dephosphorylated. E. coli ER2683 was transformed with the ligation reaction by electroporation. Transformants were pooled and the plasmid population was purified to create a primary plasmid library. A partial sample of such purified plasmid was completely digested with excess MseI restriction endonuclease and used to transform ER2683. The resulting transformant plasmid was miniprepped and analyzed by digestion with MseI restriction enzyme followed by agar gel electrophoresis. Nine plasmids examined were found to be resistant to digestion of MseI, each encoding one of two different methylase genes that function to protect DNA from cleavage by MseI. There was found. The two methylase genes were named esaDix4IM (SEQ ID NO: 3 and SEQ ID NO: 4) and esaix5IM (SEQ ID NO: 5 and SEQ ID NO: 6). Analysis of crude cell extracts prepared from such clones showed no endonuclease activity. Such methyltransferases or others may be used to protect the host's own DNA, and thus the MseI endonuclease can be successfully expressed.
[0046]
2) Sequencing the entire MseI restriction-modification system
The MseI methylase gene rather than the MseI endonuclease gene is generally obtained based on a technique called methylase selection (US Pat. No. 5,200,333) as in step 1) above. However, none of the clones obtained by methylase selection showed detectable MseI restriction endonuclease activity. One methylase clone was sequenced using an ABI 373 DNA sequencer using conventional techniques. The MseI methylase gene was identified based on amino acid homology to other N6-adenine methylases. Although the methylase clone did not produce any detectable MseI endonuclease activity, it was speculated that the endonuclease gene appears to be located adjacent to the methylase gene. Therefore, the DNA adjacent to the MseI methylase gene obtained from Micrococcus species (NEB # 446) was amplified from the genomic DNA of Micrococcus species by inverse PCR technique and sequenced.
[0047]
To locate and identify the MseI endonuclease gene, the N-terminal amino acid sequence of the highly purified MseI restriction endonuclease protein obtained from Micrococcus species was determined. MseI endonuclease may be purified from Micrococcus species (NEB # 446) as described in Example III below. An open reading frame in which the deduced amino acid sequence coincided with the N-terminal amino acid sequence of MseI endonuclease was found in the DNA sequence located 3 ′ of the methylase gene obtained by the inverse PCR technique.
[0048]
Alternatively, the N-terminus of MseI restriction endonuclease can be used to design a synonymous oligonucleotide primer for PCR amplification of a portion of the MseI endonuclease gene from Micrococcus species (NEB # 446). An amino acid sequence can be used. The resulting DNA sequence can then be used to guide inverse PCR amplification of the DNA on either side of this original portion of the MseI endonuclease gene, and the mseIM and MseI genes in this DNA sequence as described above. Can be identified. Both methods were used to clone and sequence the entire MseI restriction-modification system.
[0049]
3) Subtle optimization of MseI methyltransferase expression
To provide expression once complete genes for MseI endonuclease and MseI methyltransferase have been identified (SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 1 and SEQ ID NO: 2, respectively). They may be manipulated by various methods. Using the methylase constructs obtained above, the expression of the MseI restriction endonuclease gene was vigorously controlled under the T7 promoter control using the pET series of vectors (Madison, Wisconsin, USA, Novagen). Attempts were not made to obtain clones producing MseI restriction endonuclease.
[0050]
Introducing a second regulatable promoter before the methylase gene, using a low copy number replicon for the endonuclease gene, and increasing the copy number of the LacI repressor in the host prior to introduction of the endonuclease gene. A unique combination of methods was used to control overexpression of recombinant MseI endonuclease.
[0051]
When the cells were growing rapidly in the exponential growth phase, it was found that the methylase construct obtained by methylase selection did not completely protect the chromosomal DNA of the host E. coli. In order to enhance the expression of methylase so that mseIR is successfully introduced and expressed in cells and thus fully protect the host DNA, the methylase gene is amplified from Micrococcus species DNA and the expression of the constituent promoters at various intensities Below was cloned in the family of vectors (pNK series, see Example IV below). In this attempt, no methylase constructs were obtained for the expression of the two highest levels of promoter, and we believe that this is due to cellular toxicity due to methylase overexpression. A construct with two lower expressed promoters failed to fully protect the host against cleavage by MseI when checked in the exponential growth phase. Random mutagenesis is performed in the promoter region by error-prone PCR in one of the promoter constructs to fully protect the host DNA during rapid growth but to keep methylase expression below the level of toxicity. went. Using the methylase selection technique cited above, mutant clones expressing MseI methylase are selected, then exponentially growing cells are recovered, DNA is purified from such host cells, and MseI cleavage is performed. Individual clones were examined for their ability to fully protect host genomic DNA from MseI cleavage during rapid exponential growth by examining for complete protection of. One of the constructs found to be fully protected against MseI was then used for expression of the MseI endonuclease.
[0052]
This method of modulating methyltransferase expression to achieve complete protection at all stages of host cell growth indicates that endonucleases are difficult or impossible to express in host cells. It may be demonstrated that it is applicable to the other systems shown (see US Pat. Nos. 6,025,179 and 6,048,731).
[0053]
4) Expression of MseI restriction endonuclease under the control of an inducible promoter
In order to optimize the expression of the recombinant MseI of the present invention, inducible or constitutive promoters are well known and may be used to express the mseIR gene at high levels in recombinant hosts. Similarly, known high copy number vectors may be used to achieve high levels of expression. In accordance with the present invention, a particularly preferred method for expression of MseI restriction endonuclease is expression of MseI endonuclease during cell growth prior to induction, such as, for example, pVR-24 (Beverly, Massachusetts, USA). Is an expression vector designed to limit This plasmid contains a fragment encoding the replication function (ori), a chloramphenicol resistance gene (Cm), and a gene encoding kanamycin resistance flanked by suitable restriction endonuclease sites for cloning. The cI857 gene encodes a mutated version of the lambda bacteriophage repressor protein and conditionally binds to the DNA sequence that overlays PL and PR (lamda bacteriophage major left and right promoters, respectively). The lacI gene encodes a repressor protein, LacI, and P _T7 Conditionally binds to a DNA sequence (lac operator) that is constructed to overlay (bacteriophage T7 RNA polymerase transcription promoter). Simply put, the antisense promoter is active at high temperatures (42 ° C) in the absence of IPTG, but P _T7 Is suppressed by LacI. In the presence of IPTG at 30 ° C, expression is P _T7 (See FIGS. 14 and 15). With an intermediate concentration of IPTG at an intermediate temperature, an intermediate level of expression is obtained.
[0054]
In order to obtain stable clones that overexpress the restriction endonuclease, the host is generally pre-protected from restriction endonuclease digestion. In the present invention, MseI cleavage, such as esaDix4IM or esaDix5IM, is expressed by cloning the MseI methylase gene or in another compatible plasmid in a manner that provides complete protection against MseI cleavage. This is accomplished by cloning another methylase gene that protects against. As shown in Example 5 below, when a DNA fragment encoding a novel promoter sequence precedes the MseI methyltransferase gene, the stability of the expression plasmid containing the restriction endonuclease gene construct and / or its mRNA is improved. It turns out that it can be done. The MseI endonuclease is produced by growing a host containing the MseI endonuclease and a protective methylase gene, inducing under appropriate expression conditions, harvesting the cells, and then purifying the MseI endonuclease.
[0055]
The present invention further introduces a gene encoding an MseI restriction endonuclease and a gene encoding a methyltransferase DNA that protects host DNA from cleavage of MseI into a microorganism to grow an organism under conditions suitable for expression of the MseI endonuclease. A method for producing an MseI restriction endonuclease is provided that uses a recombinant DNA modification method to transform the microorganism to recover it and then purify the MseI endonuclease.
[0056]
While the steps outlined above represent preferred embodiments for practicing the present invention, it will be apparent to those skilled in the art that the above approach can be varied according to known techniques.
[0057]
The following examples are given to illustrate embodiments of the present invention because they are preferred in practice. Such embodiments are merely examples, and the present invention is not considered to be limited to them except in the claims of this specification.
[0058]
The references cited above and below are incorporated herein by reference.
[0059]
【Example】
Example 1: Cloning of the MseI methyltransferase gene (mseIM)
Grow Micrococcus species (NEB # 446) overnight in 1 L LB broth medium, harvest cells, and use Qiagen Genome Chip 100 / G Genomic DNA Purification Kit (Cat # 10243) according to manufacturer's instructions. Genomic DNA was isolated. The genomic DNA was partially digested with Sau3AI to produce a 1-10 kb fragment, and 20 μg of this digested genomic DNA was ligated to 3 μg of pBR322 digested with BamHI and dephosphorylated. Escherichia coli strain ER2502 was transformed with the ligation mixture. Approximately 100,000 transformants were obtained. Transformants were pooled and grown in 500 ml LB broth medium containing 100 μg / ml ampicillin and the plasmid population was purified to form the primary plasmid library. 2 μg of this plasmid library was used to transform ER2505 by complete digestion with excess MseI restriction endonuclease. The resulting transformant plasmid was selected for the second selection. Eighty transformants were obtained and 16 of these plasmid DNAs were analyzed by MseI restriction enzyme digestion followed by agar gel electrophoresis. Of the 16 plasmids examined, 14 were found to be resistant to MseI digestion and have the same mseIM gene (SEQ ID NO: 1, SEQ IDNO: 2) on an approximately 1.6 kb Sau3AI fragment. It has been found. Analysis of crude cell extracts prepared from these 14 clones showed no MseI activity.
[0060]
Example 2: Cloning of two methylase DNAs from a DNA sample from an environment protecting the DNA against cleavage by MseI endonuclease
To look for alternative methyltransferase DNA that can protect the DNA from cleavage by MseI endonuclease, a library of clones from a DNA source other than Micrococcus sp. (NEB446) contains one or more MseI restriction sites It may be constructed in a vector. This library of clones was then digested to recover one or more MseI digests to destroy unprotected clones, followed by recovery of protected clones, as in Example I above. Selection as described above by plasmid transformation. Such a library was created from DNA isolated from prokaryotic mixed green filaments and pedestal colony samples growing at 68 ° C. at Dixie Valley, Nevada Hot Springs. 2 μg of DNA was digested with NsiI endonuclease, ligated to 1 μg of vector pNEB193 previously cut with PstI and dephosphorylated. E. coli ER2683 was transformed with the ligation product by electroporation to obtain approximately 1,000,000 transformants. Transformants were pooled and grown in 500 ml LB broth medium containing 100 μg / ml ampicillin and the plasmid population was purified to form the primary plasmid library. 1 μg of this plasmid library was completely digested with excess MseI restriction endonuclease and used to transform ER2683. The resulting transformant plasmid was miniprepped and analyzed by MseI restriction enzyme digestion followed by agar gel electrophoresis. The nine plasmids tested were found to be resistant to MseI digestion and both were found to encode one of two different methylase genes that function to protect the DNA from cleavage by MseI. did. These two methylases were named esaDix4IM and esaDix5IM (SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and SEQ ID NO: 6). Analysis of crude cell extracts prepared from these clones showed no endonuclease activity. These methyltransferases or others may be used to protect the host's own DNA and therefore can successfully express the MseI endonuclease.
[0061]
Example 3: Identification and sequencing of MseI restriction endonuclease gene using N-terminal amino acid sequence and DNA sequence adjacent to MseI methylase obtained by inverse PCR method
(A) Purification of MseI restriction endonuclease derived from Micrococcus species to near homogeneity
Cells of Micrococcus species (NEB # 446) were grown in LB medium at 30 ° C. After 20 hours of growth, the cells were collected by centrifugation and stored at -70 ° C until use. All of the procedures were performed on ice or at 4 ° C. MseI endonuclease was purified according to the same scheme as Example VI. Approximately 10,000 units of MseI activity were purified to near homogeneity. 16 μl of the peak fraction was loaded on an SDS-PAGE protein gel and subjected to electrophoresis. The gel was stained with Coomassie Blue R250, and a remarkable band of about 21 kD corresponding to MseI restriction endonuclease activity was observed.
[0062]
(B) Amino terminal sequence of MseI protein
Electrophoresed MseI restriction endonuclease prepared as described and modified as previously described according to the method of Matsudaira (Matsudaira, P., J. Biol. Chem., 262: 10035-10038, 1987) And then electrically blotted (Looney, et al., Gene 80: 193-208, 1989). The membrane was stained with Coomassie Blue R-250, a protein band of approximately 21 kd was excised, and subjected to continuous degradation using a model 407A gas phase protein sequencer from PerkinElmer (Foster City, Calif., USA). (Waite-Rees, et al., J. Bacteriol., 173: 5207-5219, 1991). The first 25 residues of the 21 kD protein are (Met) -Thr-His-Glu-Pro-Thr-Asp-Asp-Pro-Asp-Phe-Ile-Val-Met-Ala-Ala-Ser-Ala-Xxx- It corresponded to Asn-Leu-Ala-Asp-Xxx-Tyr (SEQ ID NO: 10). Using this data, the endonuclease gene was identified by comparison with the amino acid sequence deduced from the DNA sequence adjacent to the methylase gene.
[0063]
(C) Determination of the DNA sequence adjacent to the mseIM methylase
Template preparation for inverse PCR amplification: 1 μg of Micrococcus species (NEB # 446) DNA in 10 × HaeII restriction endonuclease for 1 hour at 37 ° C. in 1 × NEB buffer # 4 in 50 μl reaction volume Digested. The HaeII enzyme was heat inactivated by incubating at 75 ° C. for 20 minutes. 50 μl of 10 × T4 DNA ligase buffer and 400 μl of dH ₂ Oe followed by 5 μl (2000 NEB units) of T4 DNA ligase (NEB # 202) was incubated at 16 ° C. for 16 hours to circulate the HaeII digested DNA. Next, a part of this cyclization ligation product was used as a template for the subsequent inverse PCR reaction.
[0064]
Primers MseI-IP1 and MseI-IP2 having the sequences shown below were synthesized. These primers are hybridized with the MseI endonuclease and oriented in opposite directions with respect to each other.
[0065]
Primer MseI-IP1
5'-CTTCTGCAGCCGATTTTCATAGTGAGGC-3 '
(SEQ ID NO: 11)
Primer MseI-IP2
5′-GTTCTGCAGATCCGGATCATCCGTGCGG-3 ′
(SEQ ID NO: 12)
[0066]
For reactions that succeeded in amplifying the product, a reaction mix was made by combining:
10 μl of 10 × Vent® reaction buffer
6 μl of 4 mM dNTP solution
5 μl of 10 μM concentration primer MseI-IP1
5 μl of 10 μM concentration primer MseI-IP2
3 μl of 100 mM MgSO ₄ (Final concentration of Mg ++ 5 mM)
12.5 μl of circularized DNA template (approximately 25 ng)
58 μl dH ₂ O
2 μl (4 units) of Bent® exo-polymerase NEB # 257
[0067]
PCR amplification conditions are: 1 cycle at 95 ° C for 3 minutes, followed by 4 cycles of 95 ° C for 30 seconds, 52 ° C for 30 seconds and 72 ° C for 1.5 minutes, followed by 95 ° C There were 20 cycles of 30 seconds at 62 ° C. for 30 seconds and 72 ° C. for 1.5 minutes. 10 μl of the PCR reaction was analyzed by electrophoresis on a 0.8% agar gel.
[0068]
An approximately 1350 base pair product was observed in the HaeII circular template PCR reaction. The product was purified on a gel and suspended in 25 μl of DNA (1 × TE) buffer. The PCR product was then sequenced by an ABI373 automated sequencing system according to the manufacturer's instructions using the PCR primers described above as sequencing primers. Furthermore, the MseI endonuclease region was amplified by PCR in the same reaction with the following primers, and the PCR product was sequenced.
[0069]
Primer MseI-IP3
5'-GGTTCTGCAGGTTAAGGAGGTTTAACATATACCCCACGAACCGACGGATG-3 '
(SEQ ID NO: 13)
Primer MseI-IP4
5'-GTTGGATCCGTCGACGCTTCTCCGCGTACCCAGCG-3 '
(SEQ ID NO: 14)
[0070]
The MseI endonuclease gene is identified by comparing amino acid sequence data obtained from the N-terminal amino acid sequence of the MseI endonuclease with the amino acid translation of the DNA sequence adjacent to the MseI methylase gene. An open reading frame is found that is oriented in the same direction as the MseI methylase and overlaps the methylase gene with 7 amino acid residues, in which the first 25 amino acids encoding the DNA sequence are derived from the MseI endonuclease protein. It was consistent with the determined amino acid sequence.
[0071]
The inserts with MseI methylase gene and restriction endonuclease gene were sequenced using GPS®-1 from the genome priming system (New England Biolabs, Massachusetts, USA). GPS (registered trademark) -1 contains Tn7 modified with the kptamine-resistant nptII gene, and is a member of the MseI methylase gene according to the manufacturer's instructions (New England Biolabs, Beverly, Mass., USA). Inserts were made in vitro in pVR-18 and pNEB193 containing parts. These insertions were then made using the ABI 373 automatic sequencing system using primers included in the GPS®-1 kit (primer S and primer N for the left and right ends of the transprimer, respectively) according to the manufacturer's instructions. The product sequence was determined.
[0072]
Example 4: Optimization of MseIM expression
(1) Placing the MseI methylase gene under another strength of the constituent promoter
To achieve a range of constitutive expression of MseI methylase, a related family of pNK vectors (provided by N. Keckner) containing different strength constitutive promoters was utilized. Such a plasmid contains either a WT or mutant pHis promoter upstream of the BamHI site and is a derivative of the RS415 plasmid (Simons, et al., Gene, 53: 85-96, 1987). Names and promoter strengths are as follows:
[0073]
[Table 1]

[0074]
The plasmid was digested with BamHI, MunI and BanII, and the backbone of the vector containing the constitutive promoter was gel-purified (to assist gel purification of the vector backbone by removing plasmid fragments of similar size. Including digestion).
[0075]
To prepare the MseI methylase gene for insertion downstream of the above-described constitutive promoter, Vent® DNA polymerase, 1 × Thermopol buffer, 4 mM MgSO ₄ 80 ng pVR19 plasmid (R. Vaisvila) containing the MseI methylase gene as template in a 100 μl PCR reaction, and 5′-GAACC introduced upstream of the BamHI site for the methylase gene GGATCC GACCCTGAGTGAGAACATGCCC-3 ′ (SEQ ID NO: 15) primer and 5′-AGGTCG introduced downstream of the MfeI site CAATTG PCR was performed using CCAGGGGGTCCTTCTCCGCGTAC-3 ′ (SEQ ID NO: 16) primer. 25 cycles of 95 ° C. for 10 seconds, 60 ° C. for 60 seconds and 72 ° C. for 75 seconds were performed. The resulting 1019 base pair PCR product was purified using the Chiaquick PCR purification protocol, digested sequentially with BamHI and MunI, and purified once more using the Chiaquick PCR purification protocol.
[0076]
All four BamHI-MunI vector backbones were ligated with the MseI methylase gene, transformed into ER2688 cells, and Luria-Bertani (1 gram glucose and 1 gram MgCl per liter). ₂ (Hereinafter referred to as complementary LB). However, attempts to place the MseI methylase under the two highest levels of expression failed, presumably because of instability resulting from high levels of methylation in the cells. The constructs containing two lower levels of expression, pNKR1707MseIm and pNKR2213MseIm, were restricted by MseI of plasmid DNA purified from such cells (1 μg plasmid DNA in 50 μl volume, 20 units MseI for 1 hour at 37 ° C. Complete methylation of cellular DNA did not occur, as judged by sensitivity to).
[0077]
(2) Construction of a library of constituent promoters that were randomly mutagenized by error-prone PCR
In order to find an intermediate level promoter construct for MseI methylase between pNKR2213MseIm and clearly unstable pNKR1786MseIm, the constituent promoter regions were subjected to random PCR mutagenesis and selection. In the mutagenesis protocol, high levels of Taq DNA polymerase (5 units per 100 μl reaction volume), heterogeneous dNTP pool (1.2 mM dCTP and TTP; 0.2 mM dATP and dGTP), high levels of MgCl 2 (7 mM), The presence of MnCl2 (0.5 mM), 2 ng pNKR1707 MseIm per 100 μl volume and a high cycle number (35) PCR were employed. Primers adjacent to the MseI methylase at the AgeI and BamHI restriction sites are 5′-GCGATACAGACCGGTTCAGAGGATAAAG-3 ′ (SEQ ID NO: 17) and 5′-GGTCGGATCCGGCGATACAGCGAG-3 ′ (SEQ ID NO: 18), respectively.
[0078]
After PCR, the pNKRMseIm construct with the AgeI-BamHI restriction site was cleaved off the promoter copy mutated with AgeI and BamHI, purified by gel with Qiagen gel purification kit, and purified by removing its endogenous component promoter. Connected. After electroporation into ER2688 competent cells, 20,000 colonies were obtained. Such colonies were pooled and the plasmid was purified using the Qiagen purification protocol. This constructed a library of constitutive promoters that were randomly mutagenized upstream of the MseI methylase gene.
[0079]
(3) Selection of clones that yield plasmids resistant to MseI restriction digestion
To select a plasmid that processes a mutated constitutive promoter that produces a stable, high level of methylation, expose 5 μg of the plasmid library to MseI restriction (5 μg of DNA, 50 units of MseI) at 37 ° C. for 4 hours. Followed by a 20 minute incubation at 65 ° C. to inactivate the MseI restriction endonuclease. A portion (250 ng) of the exposed pool was transformed with ER2688 calcium-competent cells, plated on complemented LB agar plates and grown overnight at 37 ° C. This produced 63 colonies.
[0080]
Six out of 63 colonies were randomly selected and further individual experiments were performed; after overnight growth in 10 ml of supplemented LB medium, using Qiagen's Qia-prep spin miniprep protocol. Plasmid DNA was purified. When 100 ng of purified plasmid DNA was exposed to 20 units of MseI at 37 ° C. for 30 minutes, all six were completely restricted, indicating an inappropriate level of methylation.
[0081]
The remaining 57 colonies were pooled and plasmid purified using Qiagen's plasmid purification protocol. From this pool of plasmids, 50 ng was subjected to a longer (overnight) 50 units of MseI exposure followed by a 20 minute incubation at 65 ° C. to inactivate the MseI restriction endonuclease. A portion (4 ng) of the exposed pool was transformed with ER2688 calcium-competent cells, plated on complemented LB agar plates and grown overnight at 37 ° C. This produced 13 colonies.
[0082]
Nine out of 13 colonies were randomly selected and further individual experiments were performed; after overnight growth and purification of the plasmid as previously described, 1 μg of plasmid DNA was 50 units in a 50 μl reaction volume. Incubation with MseI at 37 ° C. overnight revealed that 7 out of 9 were completely methylated.
[0083]
To further verify the level of methylation present in the cells, 7 colonies were recovered for plasmid purification during the logarithmic growth phase of the culture (overnight cultures were made 1: 100 in fresh supplemented LB growth medium. After 4 hours of dilution, the cells were collected at 37 ° C.). Such cells are expected to replicate DNA at a rate such that methylation by the expressed MseI methylase cannot achieve full methylation. Plasmid DNA was purified from such logarithmically growing cultures using Qiagen's Qia-prep purification protocol, and 0.5 μg of this plasmid DNA was incubated with 50 units of MseI overnight at 37 ° C. did. Using this more difficult standard for methylation, 3 out of 7 colonies were fully protected (methylated) and resistant to restriction cleavage.
[0084]
Three clones (# 4, # 9 and # 10) producing stable and complete levels of MseI methylation were mapped by AgeI and BamHI and using primers with an annealing site upstream of the promoter region And had a promoter region that was examined by sequencing (5'-GGATCTTCCAGGTGTGCATGAACG-3 '(SEQ ID NO: 19)). Two of the three clones (# 9 and # 10) are identical; therefore, the two-step selection process described results in two separate promoters that result in a stable, complete level of MseI methylation. Was found.
[0085]
Unexpectedly, both promoters # 4 and # 9 / # 10 were not mutagenized constitutive promoters as experimentally designed, but instead were contaminated with low levels of E. coli DNA present in the plasmid preparation. It was the AgeI-BamHI E. coli sequence that must have originated.
[0086]
The # 4 promoter appears to be approximately 1000 base pairs in length according to the AgeI / BamHI mapping, and according to sequencing, the first 438 base pairs are E. coli K-12MG1655 segment 349 (deposited accession number). AE000459) and base numbers 7813-8251. Upon examination of the sequence data, a BamHI site was found at base number 8814, resulting in a 1002 base pair AgeI-BamHI E. coli fragment. This E. coli sequence contains the 5 ′ end of yigW_2orf and two predicted promoters, one of which is oriented in the same direction as the MseI methylase (8672-8704th).
[0087]
The mapping of the # 9 / # 10 promoter appears to be approximately 420 base pairs in length by AgeI / BamHI; according to sequencing, the promoter is located in E. coli K-12MG1655, section 41 (deposited accession number, AE000151). ) Base numbers 2511 to 2998. This determined the 488 base pair AgeI-BamHI E. coli fragment, which contains two predicted promoters oriented in the same direction as the MseI methylase at the 5 ′ end of cof orf and base numbers 2605-2632 and 2714-2742. contains. Further investigation was performed using this # 9 / # 10 sequence.
[0088]
(4) Further optimization of MseI methylase expression
Using the above strategy, expression levels of MseI methyltransferase that allowed expression of MseI endonuclease in plasmid pVR-25 were achieved. Unexpectedly, when transformed and propagated for the first time, ER2566 host cells with MseI methylase (# 9 above) and MseI endonuclease optimized in plasmid pVR-25 expressed MseI endonuclease. When this construct was stored in glycerol at -70 ° C, MseI was not stably maintained.
[0089]
The MseI methylase construct was further modified to achieve greater modification of the host MseI. As noted above, attempts to place the MseI methylase under the highest two levels of constitutive expression failed, probably due to instability resulting from high levels of methylation in the cell. In order to achieve maximally acceptable levels of methylation, a new M.P. An MseI expression plasmid, pVR-26, was constructed. PVR-26 was constructed by inserting a second promoter derived from that described in (3) above (see Table 2). M.M. A 1.244-kb DNA fragment containing a region encoding MseI (mseIM gene) and an upstream promoter from plasmid pNKR1707mseIM-9 (digested with PmeI and MfeI), and cleaved with EcoRI and HincII P in pNEB193 (New England Biolabs, Massachusetts, USA) _lacUV5 This was done by inserting just downstream of the promoter. Another MseI methylase construct, pVR27 is P _lacUV5 It was generated by deleting the 0.379-kb PmeI-AflIII fragment containing the promoter and the lacI operator from pVR26. Stable expression of the MseI endonuclease was obtained with the pVR-26mseIM methylase expression vector.
[0090]
[Table 2]

[0091]
Example 5: Optimization of MseI restriction endonuclease expression
(1) Construction of expression vector
As is well known in the art, restriction endonucleases are cytotoxic proteins. In many cases, attempts to clone toxic genes into plasmids designed to facilitate high expression are extremely difficult. One particularly preferred plasmid for expressing cytotoxic genes is pLT7K (Kong, et al., Nucl. Acids Res., 28: 3216-3222, 2000). This plasmid contains a fragment encoding the replication function (ori), a gene encoding β-lactamase, and a gene encoding kanamycin resistance flanking a restriction endonuclease suitable for cloning. The cI857 gene is a variant derived from a lambda bacteriophage repressor protein that conditionally binds to a DNA sequence (CI operator) that overlays PL and PR (the major left and right promoters of lambda bacteriophage, respectively). Code. The lacI gene encodes the repressor protein LacI, which conditionally binds to a DNA sequence (lac operator) constructed to overlay PT7 (the bacteriophage T7 RNA polymerase transcription promoter). Simply put, at high temperatures (42 ° C) without IPTG, the antisense promoter is active and P _T7 Is suppressed by LacI. At 30 ° C with IPTG, expression is P _T7 Arise from.
[0092]
In order to adapt pLT7K for overexpression of the MseI restriction endonuclease gene, an NdeI restriction endonuclease site and a ribosome binding site were introduced. In addition, the colEI replicon was changed to a p15A replicon and the number of copies was reduced to 1/3 (from about 50 to about 15). To achieve this, pLT7K was digested with AclI and BamHI. The resulting 1.2-kb fragment containing cI857, lambda PL, Kn resistance gene and T7 promoter was isolated from the agar gel using the Qiagen Qiaquick Gel Purification Kit (Cat. It was ligated to the pACYC184-T7terΔPshAI vector digested with BamHI. pACYC184-T7terΔPshAI is a PshAI deletion derivative of pACYC184-T7ter. This construct was named pVR-24 (FIG. 14).
[0093]
Positive direction (5'-AGACTCCCC CATATG The open reading frame (ORF) for the mseIR gene was amplified by PCR with a set of primers of ACCCACGAACCGACGGATG-3 ') (SEQ ID NO: 20) and reverse direction (5'-GGGGTGCCCCTAGTAGATTTAGTAGGACCGGGG-3') (SEQ ID NO: 21). . The underlined base indicates the position of the NdeI cleavage site of the forward primer. PCR was performed using 500 ng of Micrococcus species (NEB # 446) chromosomal DNA and primers as a template in a Bent® DNA polymerase, 1 × Thermopol buffer, 100 μl PCR reaction. A cycle consisting of 95 ° C. for 15 seconds, 68 ° C. for 60 seconds, and 72 ° C. for 45 seconds was performed 25 times. The resulting 700 base pair PCR product was purified using the Chiaquick PCR purification protocol, treated with Klenow enzyme, digested with NdeI, and purified once more using the Kiaquick PCR purification protocol.
[0094]
The resulting 700 base pair NdeI-blunt end fragment containing the MseI restriction endonuclease gene was ligated to the pVR-24 vector digested with NdeI and StuI and previously modified with the MseI methylase gene construct, pNKR1707 MseIm-9. ER2502 cells were transformed with the ligation. Of the 18 transformants analyzed, 3 contained the mseIR gene. After sequencing the DNA insert containing the MseI restriction endonuclease gene, one recombinant plasmid, pVR-25, was selected to produce the MseI restriction endonuclease.
[0095]
(2) Stock construction
To increase the LacI repressor copy number in the host, strain ER2833 (T7lacIq strain) was constructed as described in US patent application Ser. No. 09 / 689,359.
[0096]
(3) Optimization of MseI restriction endonuclease overexpression in E. coli in combination with another host and a plasmid expressing MseI methylase at different levels. To optimize the overexpression of MseI restriction endonuclease in E. coli, these MseI The expression strain, ER2566 / pCEF-8, was pre-protected from autolysis of MseI endonuclease by having one of the methylase-expressing plasmids (pNKR1707MseIm-9, pCR-26 and pVR-27). Was imported. ER2566 / pCEF-8 is an inducible lac promoter and a pSYX20-based plasmid characterized by low levels of T7 lysozyme and natural inhibition of T7 RNA polymerase, under the control of pCEF-8, the gene for T7 RNA polymerase. A host strain containing a chromosomal copy. For more information, see Moffatt, B. et al. A. And Studio, F .; W. See "T7 lysozyme inhibits transcription by T7 RNA polymerase" (Cell, 49: 221-227, 1987). In uninduced cells, lysozyme reduces the basic activity of T7 RNA polymerase and increases the range of target genes that can be stably maintained in the expression host. Furthermore, another expression strain, ER2833 / pCEF-8, having one copy of the lacIq gene on the F ′ episome was used.
[0097]
Overall, 6 strains were used to study MseI restriction endonuclease expression in E. coli (Table 2). All strains contain the pVR-25 plasmid expressing the MseI restriction endonuclease and the pCEF-8 plasmid encoding the T7 bacteriophage lysozyme gene. In order to select for high yields of MseI restriction endonuclease, transformed host cells were grown using various growth conditions. The preferred medium for optimization experiments is Luria-Bertani (1 gram glucose and 1 gram MgCl per liter). ₂ (Hereinafter referred to as supplemental LB).
[0098]
Growth conditions were as follows.
MseRM1 : Cells from individual colonies were grown in 0.5 liter LB medium at 42 ° C. for 8 hours, after which IPTG was added at a final concentration of 0.2 mM to induce T7 RNA polymerase and one at 30 ° C. Cells were grown overnight (15 hours). Antibiotics were added as needed: 30 μg / ml kanamycin, 100 μg / ml ampicillin and 30 μg / ml chloramphenicol. Finally, those cultured by centrifugation were collected and frozen at -20 ° C.
[0099]
MseRM3 For each experiment, cells from individual colonies were grown overnight (17 hours) in 0.5 liter LB medium at 30 ° C., after which IPTG was added at a final concentration of 0.2 mM to add T7 RNA polymerase. Induction and cells were allowed to grow for 4 hours. Antibiotics were added as needed: 30 μg / ml kanamycin, 100 μg / ml ampicillin and 30 μg / ml chloramphenicol. Finally, those cultured by centrifugation were collected and frozen at -20 ° C.
[0100]
MseRM4, MseRM5 and MseRM6 : The bacterial culture was kept as a cryopreservation solution at −70 ° C. in 50% glycerol. The culture used for seeding was streaked on LB plates containing appropriate antibiotics to obtain a single colony. Individual colonies are resuspended in 1 ml LB medium and seeded in 1000-ml flasks containing 500 ml LB medium supplemented with 30 μg / ml kanamycin, 100 μg / ml ampicillin and 30 μg / ml chloramphenicol. did. Cells were grown overnight (16 hours) at 37 ° C. and 250 rpm in a shaker incubator. Thereafter, IPTG was added at a final concentration of 0.2 mM. The cells were further cultured for 4 hours, and then the cells were collected by centrifuging at 8,000 rpm for 5 minutes at 4 ° C. and frozen at −20 ° C.
[0101]
Two preferred restriction endonuclease assays were used to identify high level expression clones.
Ultrasonic fracturing method The induced cells (500 ml) were collected and resuspended in 20 ml of ultrasonic disruption buffer containing 10 mM Tris HCl (pH 7.5) and 1 mM EDTA. Cells were sonicated by four 30 second impacts with a macro-chip probe on ice. A portion of the crude extract was added to lambda DNA (1 μl) in NEB buffer 2 buffer (50 μl) and incubated at 37 ° C. for 1 hour. DNA was fractionated by 0.8% gel electrophoresis and visualized by EtBr staining.
[0102]
Express method Overnight, collect 1 ml of culture or induction culture (10-500 ml) and resuspend in 0.2 ml buffer containing 50 mM Tris HCl, pH 7.5 and 25% (volume / volume) sucrose. And mixed until the solution was homogeneous. 11 μl of 200 mM EDTA (pH 8.0) and freshly prepared 10 mg / ml lysozyme in 0.25 M Tris HCl (pH 8.0) were added to the 200 μl solution and incubated on ice for 5 minutes. 11.5 μl of 1M MgCl ₂ And 24.2 μl of 5% (volume / volume) Brij-58 was added. The solution was mixed gently and incubated at room temperature for 15 minutes. After incubation, the crude cell lysate was centrifuged at 4 ° C. for 15 minutes at the maximum speed of the microcentrifuge. The supernatant was pipetted into a new Eppendorf tube and stored on ice until needed. Lambda DNA substrate (1.0 μg) was digested by serial dilution of the cell extract in MseI reaction buffer (NEB buffer 2) for 1 hour at 37 ° C. DNA was fractionated by electrophoresis and visualized by EtBr staining. Activity was determined by the presence of an appropriately sized band associated with MseI digestion of lambda DNA.
[0103]
The results for optimization of MseI restriction endonuclease expression are summarized in Table 3.
[0104]
Different yields of MseI restriction endonuclease (0.08-0.5x10 depending on the MseRM1 strain) ⁶ Wet weight of U / g cells) was obtained. Cells increased slowly and the lag time was otherwise long.
[0105]
To enhance the stability and reproducibility of the lac-based recombinant expression system, lacI on the F ′ episome _q A new host strain ER2833 with one copy of the gene was constructed (US patent application, Ser. No. 09 / 689,359). In fact, lacI _q The stability of expression and plasmid maintenance in the host (MseRM3) was significantly enhanced: the yield of MseI restriction endonuclease was 0.5-1.4 × 10 ⁶ It was the wet weight of U / g cells. The MseI restriction endonuclease purified from this strain (see Example 4) was substantially free of non-specific endonuclease and exonuclease, and the final yield was about 150,000 U / g. . It was about 100 times more than that obtained from the original Micrococcus species (NEB # 446).
[0106]
[Table 3]

[0107]
Unfortunately, the MseRM3 strain did not show MseI restriction endonuclease activity after storage and recovery at -70 ° C. In order to solve this problem, the expression level of MseI methylase was increased by constructing pVR-26 and pVR-27 (Example 4 above). Such strains (MseRM4, MseRM5 and MseRM6) gave high MseI restriction endonuclease yields even after storage at −70 ° C., and MseRM4 (NEB # 1284; New England Biolabs, Massachusetts, USA). Was used for scale-up in a 100 liter production fermentor (see Example 4). The yield of MseI restriction endonuclease in this large scale fermentation is 0.5 × 10 6 per wet cell weight. ⁶ U / g.
[0108]
Example 6: Production of recombinant MseI restriction endonuclease
MseI restriction enzyme was produced from recombinant E. coli strain NEB # 1284 grown in the late logarithmic growth phase in a 100 liter fermentor. A sample of this cell was deposited with the ATCC on August 28, 2000 under the terms and conditions of the Budapest Agreement and received “ATCC Accession Number PTA-2421”.
[0109]
(A) Cell proliferation
The transformed E. coli host, NEB # 1284, containing the recombinant MseI restriction endonuclease was stored as a cryopreservation solution in 50% glycerol at -70 ° C. The culture used for seeding was streaked on LB agar plates containing ampicillin (100 μg / ml), chloramphenicol (30 μg / ml) and kanamycin (50 μg / ml) and overnight at 37 ° C. Incubation yielded a single colony. Several colonies were used to inoculate 10 ml LB medium supplemented with 30 μg / ml kanamycin, 100 μg / ml ampicillin and 30 μg / ml chloramphenicol. Cells were grown for 3 hours in a shaking incubator at 37 ° C., 250 rpm, and then for an additional 3.5 hours at 30 ° C. (to avoid overgrowth of the culture). The final corrected klet for this culture was 122 or mid log phase. This culture was used to inoculate 100 liters of LB supplemented with 30 μg / ml kanamycin, 100 μg / ml ampicillin and 30 μg / ml chloramphenicol. Fermentation was carried out for 18 hours at 30 ° C. with 2 SCFM aeration (standard cubic feet per minute) and a stirring speed of 200 rpm. The final corrected cullet was 313. From this fermentation, 331 grams of cells (wet weight) were collected by continuous flow centrifugation and stored at -70 ° C. A crude extract was prepared from 1 g of cells and the enzyme activity was estimated using the method described above (see Example 5). The yield of MseI restriction endonuclease in the crude extract is 500,000 # U / g, which is about 100 times higher than the crude extract of Micrococcus species (NEB # 446).
[0110]
(B) Purification of MseI restriction endonuclease from NEB # 1284
The following steps were all performed either on ice or at 4 ° C. Suspend 330 grams of cells in 990 ml Buffer A (0.15 M NaCl, 10 mM Tris HCl, pH 7.5, 10 mM BME, 1 mM EDTA and 5% (volume / volume) glycerol). 56 O.D. D. Until crushing 4 times with psig12K. 1150 ml of the supernatant was PEG precipitated by adding PEG 6000 at 7.5% and NaCl at 0.5 M and then incubated at 4 ° C. for 50 minutes. The PEG slurry was centrifuged at 4K for 30 minutes at 12K. 580 ml of the supernatant was diluted to 0.1 M NaCl with Buffer A without NaCl and applied to a 430 ml Heparin Hyper D column equilibrated with Buffer A. The column was washed with 1200 ml Buffer A and then a linear gradient of 4000 ml 0.1 M NaCl to 1.0 M NaCl was applied. Restriction enzyme activity eluted at 0.25 M to 0.35 M NaCl and was pooled. The heparin hyper D pool was diluted to 0.1 M NaCl with buffer A without NaCl and applied to an 88 ml PEI column equilibrated with buffer A. The column was washed with 100 ml of buffer A and a linear gradient of 1000 ml of 0.1M to 1.7M NaCl was applied. Restriction enzyme activity elutes at 0.7-0.9 M NaCl, buffer C (50 mM NaCl, 15 mM Tris pH 7.5, 10 mM BME, 0.1 mM EDTA and 5% (volume / volume). Dialyzed overnight against glycerol) and applied to a 20 ml source Q column equilibrated with buffer C. The column was washed with 40 ml of Buffer C and a linear gradient of 400 ml 0.05 M NaCl to 1.0 M NaCl was applied. Restriction enzyme activity eluted at 0.25 M to 0.35 M NaCl and was pooled. The source Q pool is dialyzed against buffer D (10 mM KPO4, pH 7.0, 0.075 M NaCl, 10 mM BME, 0.1 mM EDTA, and 5% (volume / volume) glycerol) and buffered. The solution was applied to a 20 ml heparin TSK column equilibrated with liquid D. The column was washed with 40 ml Buffer D and a linear gradient of 0.075 M to 1.0 M NaCl in 400 ml Buffer D was applied. Restriction enzyme activity eluted at 0.3-0.4 M NaCl and was pooled. BSA was added at a final concentration of 100 μg / ml. Pooled against storage buffer (20 mM Tris HCl, pH 7.5, 0.1 M EDTA, 1 mM DTT, 50 mM NaCl, 50% (volume / volume) glycerol, 200 μg / ml BSA) And dialyzed overnight. This purification method yielded 26,000,000 units of MseI restriction endonuclease. The MseI restriction endonuclease obtained from this purification was substantially free of non-specific endonucleases and exonucleases.
[0111]
The purity of the MseI restriction endonuclease preparation was checked by looking at the following criteria.
[0112]
1. Ligation: After 5-fold overdigestion of lambda DNA, more than 95% of the resulting DNA fragments were ligated with T4 DNA ligase (1-2 μM 5 ′ end concentration at 16 ° C.). Of this ligated fragment, 95% was re-cleavable.
[0113]
2. Continuous digestion: After incubation of 50 μl reaction containing 1 μg of lambda and 100 units of enzyme for 16 hours, the same band pattern of DNA bands was generated as a reaction performed for 1 hour with 1 unit of enzyme.
[0114]
3. Exonuclease activity: Less than 0.4% of radioactivity released after incubation of 100 units of enzyme for 4 hours at 37 ° C. in a 50 μl reaction containing 1 μg of sonicated 3H DNA (105 cpm / μg) It was done.
[0115]
All tests were performed with the following reaction buffer: NEB buffer 2 (50 mM NaCl, 10 mM MgCl ₂ 10 mM Tris HCl, 1 mM DTT, pH 7.9 at 25 ° C., supplemented with 100 μg / ml BSA). Unit determination: Lambda DNA substrate (1.0 μg) was digested with serially diluted MseI endonuclease for 1 hour at 37 ° C. in 50 μl of 1 × MseI reaction buffer (NEB buffer 2). DNA was fractionated by electrophoresis and visualized by EtdBr. Activity was determined by the presence of an appropriately sized band related to MseI digestion of lambda DNA. One unit of restriction endonuclease activity is defined as the amount required to completely digest 1 μg of substrate DNA in a total reaction volume of 50 μl in 1 hour using the specified NEB buffer.
[0116]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows a restriction map of the recombinant plasmid pVR18 encoding the MseI DNA methyltransferase gene.
FIG. 2 shows a restriction map of the recombinant plasmid pEsaDix4I encoding the putative DNA methyltransferase gene.
FIG. 3 shows a restriction map of the recombinant plasmid pEsaDix5I encoding the putative DNA methyltransferase gene.
FIG. 4 shows an agar gel analysis of the sensitivity of the pEsaDix4I plasmid encoding the putative DNA methyltransferase gene to MseI. Lane 1, uncleaved pEsaDix4I; lane 2, pEsaDix4I after overnight incubation with 10 units of MseI; lane 3, uncleaved pUC19; lane 4, pUC19 after overnight incubation with 10 units of MseI; Lane 4, pEsaDix4I + pUC19 after overnight incubation with 10 units of MseI; Lane 5, molecular weight standard (1 kb DNA ladder, New England Biolabs).
FIG. 5 shows an agar gel analysis of the sensitivity of pEsaDix5I plasmid encoding the putative DNA methyltransferase gene to MseI. Lane 1, uncleaved pEsaDix5I; lane 2, pEsaDix5I after overnight incubation with 10 units of MseI; lane 3, uncleaved pUC19; lane 4, pUC19 after overnight incubation with 10 units of MseI; Lane 4, pEsaDix5I + pUC19 after overnight incubation with 10 units of MseI; Lane 5, molecular weight standard (1 kb DNA ladder, New England Biolabs).
FIG. 6 shows the DNA sequence of the mseIM gene (SEQ ID NO: 1) and the amino acid sequence encoded thereby (SEQ ID NO: 2).
FIG. 7 shows the DNA sequence of the esaDix4IM gene (SEQ ID NO: 3) and the amino acid sequence encoded thereby (SEQ ID NO: 4).
FIG. 8 shows the DNA sequence of the esaDix5IM gene (SEQ ID NO: 5) and the amino acid sequence encoded thereby (SEQ ID NO: 6).
FIG. 9 shows the DNA sequence of the mseIR gene (SEQ ID NO: 7) and the amino acid sequence encoded thereby (SEQ ID NO: 8).
FIG. 10 shows a restriction map of the recombinant plasmid pNKR1707mseIM used to construct a library of constituent promoters randomly mutagenized by error-prone PCR.
FIG. 11 shows a restriction map of the recombinant plasmid pNKR1707mseIM-9 encoding the MseI DNA methyltransferase gene and upstream regulatory elements.
FIG. 12 shows the DNA sequence upstream of the MseI DNA methyltransferase gene (SEQ ID NO: 9) containing the optimal promoter sequence.
FIG. 13 shows the construction of plasmids pVR-26 and pVR-27 used for the controlled expression of the MseI DNA methyltransferase gene.
FIG. 14 shows the construction of pVR-24 expression vector.
FIG. 15 shows a restriction map of pVR-25 encoding the MseI restriction endonuclease gene.
FIG. 16 shows a restriction map of pCEF-8 encoding the T7 lysozyme gene.
FIG. 17 shows the mechanism of action of a strict regulatory system in pVR-25 for cloning genes encoding cytotoxic proteins.
FIG. 18 shows an assay for MseI restriction endonuclease activity in crude cell extracts made from E. coli MseRM4, MseRM5 and MseRM6. Growth conditions are described in Example 6.
FIG. 19 shows assay for MseI restriction endonuclease activity in crude cell extracts made from E. coli strain MseRM4 (NEB # 1284) after growth in a 100 liter fermentor.

Claims (7)

An isolated DNA encoding an MseI restriction endonuclease comprising the amino acid sequence set forth in SEQ ID NO: 8 . The DNA according to claim 1, comprising the base sequence represented by SEQ ID NO: 7. A recombinant DNA vector comprising DNA encoding the MseI restriction endonuclease according to claim 1 or 2 . A transformed cell transformed with the vector according to claim 3 . 5. The transformation according to claim 4, further comprising a recombinant DNA vector comprising a DNA encoding a methyltransferase comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. cell. The transformed cell according to claim 4 or 5, which is ATCC accession number PTA-2421 strain. A method for producing an MseI restriction endonuclease, comprising culturing the transformed cell according to any one of claims 4 to 6 under conditions suitable for expression of the MseI restriction endonuclease.

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